Conformationally constrained backbone cyclized peptide analogs

ABSTRACT

Novel backbone cyclized peptide analogs are formed by means of bridging groups attached via the alpha nitrogens of amino acid derivatives to provide novel non-peptidic linkages. Novel building units disclosed are N α (ω-functionalized) amino acids constructed to include a spacer and a terminal functional group. One or more of these N α (ω-functionalized) amino acids are incorporated into a peptide sequence, preferably during solid phase peptide synthesis. The reactive terminal functional groups are protected by specific protecting groups that can be selectively removed to effect either backbone-to-backbone or backbone-to-side chain cyclizations. The invention is specifically exemplified by backbone cyclized bradykinin antagonists having biological activity. Further embodiments of the invention are somatostatin analogs having one or two ring structures involving backbone cyclization.

This is a continuation of application Ser. No. 08/488,159, filed Jun. 7,1995 now U.S. Pat. No. 5,811,392.

FIELD OF THE INVENTION

The present invention relates to conformationally constrained N^(α)backbone-cyclized peptide analogs cyclized via novel non-peptidiclinkages, to novel N^(α),ω-functionalized amino acid building units, toprocesses for the preparation of these backbone cyclized peptides andbuilding units, to methods for using these peptide analogs and topharmaceutical compositions containing same.

BACKGROUND OF THE INVENTION

Peptidomimetics

As a result of major advances in organic chemistry and in molecularbiology, many bioactive peptides can now be prepared in quantitiessufficient for pharmacological and clinical utilities. Thus in the lastfew years new methods have been established for the treatment andtherapy of illnesses in which peptides have been implicated. However,the use of peptides as drugs is limited by the following factors: a)their low metabolic stability towards proteolysis in thegastrointestinal tract and in serum; b) their poor absorption after oralingestion, in particular due to their relatively high molecular mass orthe lack of specific transport systems or both; c) their rapid excretionthrough the liver and kidneys; and d) their undesired side effects innon-target organ systems, since peptide receptors can be widelydistributed in an organism.

Moreover, with few exceptions, native peptides of small to medium size(less than 30-50 amino acids) exist unordered in dilute aqueous solutionin a multitude of conformations in dynamic equilibrium which may lead tolack of receptor selectivity, metabolic susceptibilities and hamperattempts to determine the biologically active conformation. If a peptidehas the biologically active conformation per se, i.e., receptor-boundconformation, then an increased affinity toward the receptor isexpected, since the decrease in entropy on binding is less than that onthe binding of a flexible peptide. It is therefore important to strivefor and develop ordered, uniform and biologically active peptides.

In recent years, intensive efforts have been made to developpeptidomimetics or peptide analogs that display more favorablepharmacological properties than their prototype native peptides. Thenative peptide itself, the pharmacological properties of which have beenoptimized, generally serves as a lead for the development of thesepeptidomimetics. However, a major problem in the development of suchagents is the discovery of the active region of a biologically activepeptide. For instance, frequently only a small number of amino acids(usually four to eight) are responsible for the recognition of a peptideligand by a receptor. Once this biologically active site is determined alead structure for development of peptidomimetic can be optimized, forexample by molecular modeling programs.

As used herein, a “peptidomimetic” is a compound that, as a ligand of areceptor, can imitate (agonist) or block (antagonist) the biologicaleffect of a peptide at the receptor level. The following factors shouldbe considered to achieve the best possible agonist peptidomimetic a)metabolic stability, b) good bioavailability, c) high receptor affinityand receptor selectivity, and d) minimal side effects.

From the pharmacological and medical viewpoint it is frequentlydesirable to not only imitate the effect of the peptide at the receptorlevel (agonism) but also to block the receptor when required(antagonism). The same pharmacological considerations for designing anagonist peptidomimetic mentioned above hold for designing peptideantagonists, but, in addition, their development in the absence of leadstructures is more difficult. Even today it is not unequivocally clearwhich factors are decisive for the agonistic effect and which are forthe antagonistic effect.

A generally applicable and successful method recently has been thedevelopment of conformationally restricted peptidomimetics that imitatethe receptor-bound conformation of the endogenous peptide ligands asclosely as possible (Rizo and Gierasch, Ann. Rev. Biochem., 61:387,1992). Investigations of these types of analogs show them to haveincreased resistance toward proteases, that is, an increase in metabolicstability, as well as increased selectivity and thereby fewer sideeffects (Veber and Friedinger, Trends Neurosci., p. 392, 1985).

Once these peptidomimetic compounds with rigid conformations areproduced, the most active structures are selected by studying theconformation-activity relationships. Such conformational constraints caninvolve short range (local) modifications of structure or long range(global) conformational restraints (for review see Giannis and Kolter,Angew. Chem. Int. Ed. Engl. 32:1244, 1993).

Conformationally Constrained Peptides

Bridging between two neighboring amino acids in a peptide leads to alocal conformational modification, the flexibility of which is limitedin comparison with that of regular dipeptides. Some possibilities forforming such bridges include incorporation of lactams and piperazinones.γ-Lactams and δ-lactams have been designed to some extent as “turnmimetics”; in several cases the incorporation of such structures intopeptides leads to biologically active compounds.

Global restrictions in the conformation of a peptide are possible bylimiting the flexibility of the peptide strand through cyclization(Hruby et al., Biochem. J., 268:249, 1990). Not only does cyclization ofbioactive peptides improve their metabolic stability and receptorselectivity, cyclization also imposes constraints that enhanceconformational homogeneity and facilitates conformational analysis. Thecommon modes of cyclization are the same found in naturally occurringcyclic peptides. These include side chain to side chain cyclization orside chain to end-group cyclization. For this purpose, amino acid sidechains that are not involved in receptor recognition are connectedtogether or to the peptide backbone. Another common cyclization is theend-to-end cyclization.

Three representative examples are compounds wherein partial structuresof each peptide are made into rings by linking two penicillamineresidues with a disulfide bridge (Mosberg et al., P.N.A.S. US, 80:5871,1983), by formation of an amide bond between a lysine and an aspartategroup (Charpentier et al., J. Med. Chem. 32:1184, 1989), or byconnecting two lysine groups with a succinate unit (Rodriguez et al.,Int. J. Pept. Protein Res. 35:441, 1990). These structures have beendisclosed in the literature in the case of a cyclic enkephalin analogwith selectivity for the δ-opiate receptor (Mosberg et al., ibid.); oras agonists to the cholecystokinin B receptor, found largely in thebrain (Charpentier et al., ibid., Rodriguez et al., ibid.).

The main limitations to these classical modes of cyclization are thatthey require substitution of amino acid side chains in order to achievecyclization.

Another conceptual approach to the conformational constraint of peptideswas introduced by Gilon, et al., (Biopolymers, 31:745, 1991) whoproposed backbone to backbone cyclization of peptides. The theoreticaladvantages of this strategy include the ability to effect cyclizationvia the carbons or nitrogens of the peptide backbone without interferingwith side chains that may be crucial for interaction with the specificreceptor of a given peptide. While the concept was envisaged as beingapplicable to any linear peptide of interest, in point of fact thelimiting factor in the proposed scheme was the availability of suitablebuilding units that must be used to replace the amino acids that are tobe linked via bridging groups. The actual reduction to practice of thisconcept of backbone cyclization was prevented by the inability to deviseany practical method of preparing building units of amino acids otherthan glycine (Byk et al., J. Org. Chem., 587:5687, 1992). While analogsof other amino acids were attempted the synthetic method used wasunsuccessful or of such low yield as to preclude any generalapplicability.

In Gilon, EPO Application No. 564,739 A2; and J. Org. Chem., 57:5687,1992, two basic approaches to the synthesis of building units aredescribed. The first starts with the reaction of a diamine with ageneral α bromo acid. Selective protection of the ω amine and furtherelaborations of protecting groups provides a building unit, suitable forBoc chemistry peptide synthesis. The second approach starts withselective protection of a diamine and reaction of the product withchloroacetic acid to provide the protected glycine derivative, suitablefor Fmoc peptide synthesis.

Both examples deal with the reaction of a molecule of the general typeX—CH(R)—CO—OR′ (wherein X represents a leaving group which, in theexamples given, is either Br or Cl) with an amine which replaces the X.The amine bears an alkylidene chain which is terminated by anotherfunctional group, amine in the examples described, which may or may notbe blocked by a protecting group.

In all cases the α nitrogen of the end product originates in themolecule which becomes the bridging chain for subsequent cyclization.This approach was chosen in order to take advantage of the highersusceptibility to nucleophilic displacement of a leaving group next to acarboxylic group.

In a molecule where R is different than hydrogen there is a hightendency to eliminate HX under basic conditions. This side reactionreduces the yield of Gilon's method to the point where it is impracticalfor production of building units based on amino acids other thanglycine. The diamine nitrogen is primary while the product contains asecondary nitrogen, which is a better nucleophile. So while the desiredreaction may be sluggish, and require the addition of catalysts, theproduct may be contaminated with double alkylation products. There is nomention of building units with end group chemistries other thannitrogen, so the only cyclization schemes possible are backbone to sidechain and backbone to C terminus.

Applications of Conformationally Constrained Peptides

Conformationally constrained peptides find many pharmacological uses.Somatostatin is a cyclic tetradecapeptide found both in the centralnervous system and in peripheral tissues. It was originally isolatedfrom mammalian hypothalamus and identified as an important inhibitor ofgrowth hormone secretion from the anterior pituitary. Its multiplebiological activities include inhibition of the secretion of glucagonand insulin from the pancreas, regulation of most gut hormones andregulation of the release of other neurotransmitters involved in motoractivity and cognitive processes throughout the central nervous system(for review see Lamberts, Endocrine Rev., 9:427, 1988).

Natural somatostatin (also known as Somatotropin Release InhibitingFactor, SRIF) of the following structure:

H-Ala¹-Gly²-Cys³-Lys⁴-Asn⁵-Phe⁶-Phe⁷-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹-Thr¹²-Ser¹³-Cys¹⁴-OH

was first isolated by Guillemin and colleagues (Brazeau et al. Science,179:78, 1973). In its natural form, it has limited use as a therapeuticagent since it exhibits two undesirable properties: poor bioavailabilityand short duration of action. For this reason, great efforts have beenmade during the last two decades to find somatostatin analogs that willhave superiority in either potency, biostability, duration of action orselectivity with regard to inhibition of the release of growth hormone,insulin or glucagon.

Structure-activity relation studies, spectroscopic techniques such ascircular dichroism and nuclear magnetic resonance, and molecularmodeling approaches reveal the following: the conformation of the cyclicpart of natural somatostatin is most likely to be an antiparallelβ-sheet; Phe⁶ and Phe¹¹ play an important role in stabilizing thepharmacophore conformation through hydrophobic interactions between thetwo aromatic rings; the four amino acids Phe⁷-Trp⁸-Lys⁹-Thr¹⁰ which arespread around the β-turn in the antiparallel β-sheet are essential forthe pharmacophore; and (D)Trp⁸ is almost always preferable to (L)Trp⁸.

Nevertheless, a hexapeptide somatostatin analog containing these fouramino acids anchored by a disulfide bridge:

is almost inactive both in vitro and in vivo, although it has theadvantage of the covalent disulfide bridge which replaces the Phe⁶-Phe¹¹hydrophobic interactions in natural somatostatin.

Four main approaches have been attempted in order to increase theactivity of this hexapeptide somatostatin analog. (1) Replacing thedisulfide bridge by a cyclization which encourages a cis-amide bond, orby performing a second cyclization to the molecule yielding a bicyclicanalog. In both cases the resultant analog has a reduced number ofconformational degrees of freedom. (2) Replacing the original aminoacids in the sequence Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with more potent aminoacid analogs, such as replacing Phe⁷ with Tyr⁷ and Thr¹⁰ with Val¹⁰. (3)Incorporating additional structural elements from natural somatostatinwith the intention that these new elements will contribute to theinteraction with the receptor. (4) Eliminating one of the four aminoacids Phe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰ with the assumption that such analogswould be more selective.

The somatostatin analog, MK-678:

cyclo(N-Me-Ala⁷-Tyr⁷-(D)Trp⁸-Lys⁹-Val¹⁰-Phe)

is an example of a highly potent somatostatin analog designed using thefirst three approaches above (Lymangrover, et al., Life Science, 34:371,1984). In this hexapeptide analog, a cis-amide bond is located betweenN-Me-Ala and Phe¹¹, Tyr⁷ and Val¹⁰ replace Phe⁷ and Thr¹⁰ respectively,and Phe¹¹ is incorporated from natural somatostatin.

Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and4,235,886) includes octreotide:

the only somatostatin analog currently available. It was developed usingthe third approach described above. Here, (D)Phe⁵ and the reducedC-terminal Thr¹²-CH₂OH are assumed to occupy some of the conformationalspace available to the natural Phe⁶ and Thr¹², respectively.

The compound TT2-32:

is closely related to octreotide and is an example of implementing thefourth approach described above. The Lack of Thr¹⁰ is probablyresponsible for its high selectivity in terms of antitumor activity.

These examples of highly potent somatostatin analogs indicate that thephenylalanines in positions 6 and 11 not only play an important role instabilizing the pharmacophore conformation but also have a functionalrole in the interaction with the receptor. It is still an open questionwhether one phenylalanine (either Phe⁶ or Phe¹¹) is sufficient for theinteraction with the receptor or whether both are needed.

It is now known that the somatostatin receptors constitute a family offive different receptor subtypes (Bell and Reisine, Trends Neurosci.,16, 34-38, 1993), which may be distinguished on the basis of theirtissue specificity and/or biological activity. Somatostatin analogsknown in the art may not provide sufficient selectivity or receptorsubtype selectivity, particularly as anti-neoplastic agents (Reubi andLaissue, TIPS, 16, 110-115, 1995).

Symptoms associated with metastatic carcinoid tumors (flushing anddiarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas(watery diarrhea) are treated with somatostatin analogs. Somatostatinhas been also approved for the treatment of severe gastrointestinalhemorrhages. Somatostatin may also be useful in the palliative treatmentof other hormone-secreting tumors (e.g., pancreatic islet-cell tumorsand acromegaly) and hormone dependent tumors (e.g., chondrosarcoma andosteosarcoma) due to its anti-secretory activity.

Another important peptide, Bradykinin, is a naturally occurringnonapeptide, Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹, formed andreleased from precursors in the blood in response to inflammatorystimuli. Elevated levels of bradykinin also appear in other body fluidsand tissues in pathological states such as asthma, septic shock andcommon cold. No clinical abnormalities have been associated so far withbradykinin deficiency which indicates that bradykinin may not play acritical role in normal physiology.

However, bradykinin mediates its physiological activities by binding toa specific receptive molecule called the bradykinin receptor. Two suchbradykinin receptors have been identified so far (these are called B1and B2 receptors). Subsequent to binding, the bradykinin signaltransduction pathway includes production of prostaglandins andleukotrienes as well as calcium activation. Through these mediators,bradykinin is involved in pain, inflammation, allergic reactions andhypotension. Therefore, a substance that can block the ability ofbradykinin to bind to its receptor, namely a bradykinin antagonist,should have a significant therapeutic value for one of the followingindications: asthma, inflammation, septic shock, pain, hypotension andallergy.

The analog used herein to exemplify backbone cyclization is:

D-Arg⁰-Arg-R¹-Hyp³-Gly-Phe-R²-D-Phe-Phe⁷-Arg

(wherein, R¹ is Pro, R² is Ser in native bradykinin). The change ofproline at position 7 of native bradykinin to D-Phe confers antagonistactivity. This compound was described in Steranka, et al., P.N.A.S.U.S., 85:3245-3249, 1988 and is one of a plethora of candidate sequencesfor modification by the current technology, i.e. backbone cyclization.In this regard, it is worth noting the applications: WO 89/01781,EP-A-0370453 and EP-A-0334244 which disclose a wide range of candidatestructures. Antagonist peptides on which stability and/or tissueselectivity can be conferred by appropriate cyclization will be selectedfrom the many such known sequences.

According to the present invention a novel synthetic approach isdisclosed providing N^(α)(ω(functionalized)alkylene) amino acid buildingunits that can be used to synthesize novel N^(α)-backbone cyclizedpeptide analogs such as, but not limited to, novel somatostatin andbradykinin analogs. None of the above-mentioned references teaches orsuggests N^(α)-(ω(functionalized)alkylene) amino acids or the novelN^(α)-backbone cyclized peptide analogs of the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide backbone cyclizedpeptide analogs that comprise peptide sequences which incorporate atleast two building units, each of which contains one nitrogen atom ofthe peptide backbone connected to a bridging group as described below.In the present invention, one or more pairs of the building units isjoined together to form a cyclic structure. Thus, according to oneaspect of the present invention, backbone cyclized peptide analogs areprovided that have the general Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 orzero; d, e, and f each independently designates an integer from 1 to 10;(AA) designates an amino acid residue wherein the amino acid residues ineach chain may be the same or different; E represents a hydroxyl group,a carboxyl protecting group or an amino group, or CO—E can be reduced toCH₂—OH; R, R′, R″, and R′″ each designates an amino acid side-chain suchas H, CH₃, etc., optionally bound with a specific protecting group; andthe lines independently designate a bridging group of the Formula: (i)—X—M—Y—W—Z—; or (ii) —X—M—Z— wherein: one line may be absent; M and Ware independently selected from the group consisting of disulfide,amide, thioether, thioesters, imines, ethers and alkenes; and X, Y and Zare each independently selected from the group consisting of alkylene,substituted alkylene, arylene, homo- or hetero-cycloalkylene andsubstituted cycloalkylene.

In certain preferred embodiments, the CO—E group of Formula (I) isreduced to a CH₂OH group.

Another embodiment of the present invention involves N-backbone to sidechain cyclized peptides of the general formula (II):

wherein the substituents are as defined above.

A preferred embodiment of the present invention involves the backbonecyclized peptide analog of Formulae I or II wherein the line designatesa bridging group of the Formula: —(CH₂)_(x)—M—(CH₂)_(y)—W—(CH₂)_(z)—wherein M and W are independently selected from the group consisting ofdisulfide, amide, thioether, thioesters, imines, ethers and alkenes; xand z each independently designates an integer from 1 to 10, and y iszero or an integer of from 1 to 8, with the proviso that if y is zero, Wis absent.

Further preferred are backbone cyclized peptide analogs of the Formula Ior II wherein R and R′ are other than H, such as CH₃, (CH₃)₂CH—,(CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—,NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—,NH₂(CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, andmethylimidazole.

A more preferred embodiment of the present invention is directed tobackbone cyclization to stabilize the β-turn conformation of bradykininanalogs of the general Formula (III):

wherein M is an amide bond, x and z are each independently an integer of1 to 10, and K is H or an acyl group.

Also more preferred are backbone cyclized peptide analogs of the presentinvention comprising bradykinin analogs of the general Formula (IVa):

wherein M is an amide bond, x and z are each independently an integer of1 to 10, K is H or an acyl group, and R⁶ is Gly or Ser; or the generalFormula (IVb):

wherein x is an integer of 1 to 10; K is H or an acyl group; (R⁶) isselected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z isaccording to the amino acid specified: 1 in case of D and L-Asp, and 2in the case of D and L Glu.

Further more preferred backbone cyclized peptide analogs according tothe present invention having bradykinin antagonist activity have theFormula (V):

wherein M is an amide bond, x and z are each independently an integer of1 to 10, and K is H or an acyl group.

Specifically preferred backbone cyclized peptide analogs of the presentinvention are:

1)Ada-(D)Arg-Arg-cyclo(N^(α)(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;

2)H-D-Arg-Arg-cyclo(N^(α)(1-(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH;and

3)H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl)Gly-Hyp-Phe-N^(α)(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.

Another preferred aspect of the present invention is directed tobackbone cyclization to generate novel somatostatin analogs linkedbetween positions 6 and 11, leaving the phenylalanine side chainsuntouched. This conformational stabilization is much more rigid than thePhe⁶, Phe¹¹ hydrophobic interaction in natural somatostatin and is morestable to reduction/oxidation reactions than the Cys-Cys disulfidebridge. In other words, for the first time a stable covalent bridge canbe achieved while either one or both of the original Phe⁶ and Phe¹¹ areretained.

Moreover, backbone cyclizations can also be used to anchor the β-turn,not only in positions 6 and 11 but also inside the active reaction ofPhe⁷-(D)Trp⁸-Lys⁹-Thr¹⁰, yielding either a monocyclic analog with apreferable conformation or a very rigid bicyclic analog. Here again, theside chains of the pharmacologically active amino acids remain untouchedand the only change is in limiting the conformational space.

As used herein and in the claims in the following more preferredbackbone cyclized peptide analogs, the superscript numbers following theamino acids refer to their position numbers in the native Somatostatin.

A more preferred backbone cyclized peptide novel analog is the Formula(XIVa):

with a most preferred analog being the Formula (XIVb):

wherein m and n are 1, 2 or 3; X is CH₂OH or CONH₂; R⁵ is absent or isGly, (D)- or (L)-Ala, Phe, Nal and β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R⁷ is Phe or Tyr; R¹⁰ is absent oris Gly, Abu, Thr or Val; R¹² is absent or is Thr or Nal, and Y² isselected from the group consisting of amide, disulfide, thioether,imines, ethers and alkenes. In these monocyclic somatostatin analogs, abackbone cyclization replaces the Cys⁶-Cys¹¹ disulfide bridge, leavingthe phenylalanine side chains as in the natural somatostatin. Still morepreferred is the analog wherein Phe⁷ is replaced with Tyr⁷ and Thr¹⁰ isreplaced with Val¹⁰.

Other more preferred monocyclic analogs that anchor the molecule inpositions inside the active region rather than in positions 6 and 11 areformulae XV (a and b) and XVI (a-c):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or CONH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or (L)-Phe;R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or (L)-Phe; R¹² isabsent or is Thr or Nal, and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imines, ethers and alkenes.

Still other more preferred analogs incorporate backbone cyclization inpositions 6 and 11 as in Formula XIV, together with the backbonecyclizations as in Formula XV and XVI, yielding rigid bicyclic analogsof the Formulae XVII (a and b) and XVIII (a and b):

wherein i, j, m and n are independently 1, 2 or 3; X is CH₂OH or CONH₂;R⁵ is absent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R¹⁰ is absent or is Gly, Abu, Valor Thr; R¹² is absent or is Thr or Nal; and Y¹ and Y² are independentlyselected from the group consisting of amide, disulfide, thioether,imines, ethers and alkenes.

Other more preferred bicyclic analogs differ from Formulae XVII andXVIII by the replacement of the amino acids at positions 6 and 11 bycysteines which form a disulfide bond, leaving only one backbonecyclization in the Formulae XIX (a and b) and XX (a and b):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or Phe; R¹⁰ is absent or is Gly, Abu or Thr; R¹² isabsent or is Thr or Nal; and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imines, ethers and alkenes.

Another aspect of the present invention is a method for the preparationof cyclic peptides of the general Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 orzero; d, e, and f each independently designates an integer from 1 to 10;(AA) designates an amino acid residue wherein the amino acid residues ineach chain may be the same or different; E represents a hydroxyl group,a carboxyl protecting group or an amino group, or CO—E can be reduced toCH₂—OH; R, R′, R″, and R′″ each designates an amino acid side-chainoptionally bound with a specific protecting group; and the linesdesignate a bridging group of the Formula:

(i) —X—M—Y—W—Z—; or (ii) —X—M—Z—

wherein: one line may be absent; M and W are independently selected fromthe group consisting of disulfide, amide, thioether, thioesters, imines,ethers and alkenes; and X, Y and Z are each independently selected fromthe group consisting of alkylene, substituted alkylene, arylene, homo-or hetero-cycloalkylene and substituted cycloalkylene. This methodcomprises the steps of incorporating at least one N^(α)-ω-functionalizedderivative of amino acids of Formula (VI):

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain, optionally bound with aspecific protecting group; B is a protecting group selected from thegroup consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls;and G is a functional group selected from the group consisting ofamines, thiols, alcohols, carboxylic acids and esters, aldehydes,alcohols and alkyl halides; and A is a specific protecting group of G;into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence or with another ω-functionalized amino acid derivative.

A further object of the present invention is directed to building unitsknown as a N^(α)-ω-functionalized derivatives of the general Formula(VI) of amino acids which are prerequisites for the cyclization process:

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, optionally boundwith a specific protecting group; B is a protecting group selected fromthe group consisting of alkyloxy, substituted alkyloxy, or aryloxycarbonyls; and G is a functional group selected from the groupconsisting of amines, thiols, alcohols, carboxylic acids and esters,aldehydes and alkyl halides; and A is a protecting group thereof.

Preferred building units are the ω-functionalized amino acid derivativeswherein X is alkylene; G is a thiol group, an amine group or a carboxylgroup; R is phenyl, methyl or isobutyl; with the proviso that when G isan amine group, R is other than H.

Further preferred are ω-functionalized amino acid derivatives wherein Ris protected with a specific protecting group.

More preferred are ω-functionalized amino acid derivatives of theFormulae:

wherein X, R, A and B are as defined above.

Specifically preferred ω-functionalized amino acid derivatives includethe following:

1) N^(α)-(Fmoc)(3-Boc-amino propylene)-(S)Phenylalanine;

2) N^(α)-(Fmoc)(3-Boc-amino propylene)-(R)Phenylalanine;

3) N^(α)-(Fmoc)(4-Boc-amino butylene)-(S)Phenylalanine;

4) N^(α)-(Fmoc)(3-Boc-amino propylene)-(S)Alanine;

5) N^(α)-(Fmoc)(6-Boc-amino hexylene)-(S)Alanine;

6) N^(α)-(Fmoc)(3-Boc-amino propylene)-(R)Alanine;

7) N^(α)-(2-(benzylthio)ethylene)glycine ethyl ester;

8) N^(α)-(2-(benzylthio)ethylene)(S)leucine methyl ester;

9) N^(α)-(3-(benzylthio)propylene)(S)leucine methyl ester:

10) Boc-N^(α)-(2-(benzylthio)ethylene)glycine;

11) Boc-N^(α)-(2-(benzylthio)ethylene)(S)phenylalanine;

12) Boc-N^(α)-(3-(benzylthio)propylene)(S)phenylalanine;

13) Boc-L-phenylalanyl-N^(α)-(2-(benzylthio)ethylene)glycine-ethylester;

14) Boc-L-phenylalanyl-N^(α)-(2-(benzylthio)ethylene)-(S)phenylalaninemethyl ester;

15) N^(α)(Fmoc)-(2-t-butyl carboxy ethylene)glycine;

16) N^(α)(Fmoc)-(3-t-butyl carboxy propylene)glycine;

17) N^(α)(Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;

18) N^(α)(Fmoc)(2-Boc amino ethylene)glycine;

19) N^(α)(Fmoc)(3-Boc amino propylene)glycine;

20) N^(α)(Fmoc)(4-Boc amino butylene)glycine; and

21) N^(α)(Fmoc)(6-Boc amino hexylene)glycine.

Novel, practical, generally applicable processes for the preparation ofthese N^(α)-ω-functionalized derivatives of amino acids are a furtheraspect of this invention.

As such, an object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; A and B are protecting groups selected from the group consistingof alkyloxy, substituted alkyloxy, or aryloxy carbonyls;

comprising the steps of:

i) reacting a diamine compound of the general Formula:

wherein A, B and X are as defined above,

with a triflate of Formula CF₃SO₂—O—CH(R)—CO—E wherein E is a carboxylprotecting group and R is as defined above; to yield a compound ofFormula:

wherein A, B, E, R and X are as defined above

ii) and deprotecting the carboxyl to yield an N^(α)ω-functionalizedamino acid derivative, wherein the ω-functional group is an amine.

A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

where B is a protecting group selected from the group of substitutedalkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the sidechain of an amino acid, such as H, CH₃, etc.; X is a spacer groupselected from the group of alkylene, substituted alkylene, arylene,cycloalkylene or substituted cycloalkylene; and A is a protecting groupselected from the group of alkyl or substituted alkyl, thio ethers oraryl or substituted aryl thio ethers;

comprising the steps of:

i) reacting a compound of the general Formula B—NH—X—S—A with a triflateof the general Formula CF₃SO₂—O—CH(R)—CO—E wherein E is a carboxylprotecting group and A, X and R are as defined above, to give a compoundof the Formula:

ii) selectively removing the protecting group E, and

iii) protecting the free amino group to yield an N^(α)(ω-functionalized)amino acid derivative, wherein the ω-functional group is a thiol.

A further object of this invention is a method of making anω-functionalized amino acid derivative of the general Formula:

where B is a protecting group selected from the group of alkyloxy,substituted alkyloxy, or aryloxy carbonyls; R is the side chain of anamino acid, such as H, CH₃, etc.; X is a spacer group selected from thegroup of alkylene, substituted alkylene, arylene, cycloalkylene orsubstituted cycloalkylene; and A is a protecting group selected from thegroup of alkyl or substituted alkyl, esters, or thio esters orsubstituted aryl esters or thio esters;

comprising the steps of:

i) reacting a compound of the general Formula B—NH—X—CO—A with atriflate of the general Formula CF₃SO₂—O—CH(R)—CO—E wherein E is acarboxyl protecting group and A, B, X and R are as defined above, togive a compound of Formula:

ii) and selectively removing protecting group E, to yield anN^(α)(ω-functionalized) amino acid derivative, wherein the ω-functionalgroup is a carboxyl.

A further aspect of this invention is to provide methods for thepreparation of novel backbone cyclic peptides, comprising the steps ofincorporating at least one N^(α)-ω-functionalized derivatives of aminoacids into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence, or with another ω-functionalized amino acidderivative.

Backbone cyclized analogs of the present invention may be used aspharmaceutical compositions and for methods for the treatment ofdisorders including: acute asthma, septic shock, brain trauma and othertraumatic injury, post-surgical pain, all types of inflammation,cancers, endocrine disorders and gastrointestinal disorders.

Therefore, further objects of the present invention are directed topharmaceutical compositions comprising pharmacologically active backbonecyclized peptide agonists and antagonists prepared according to themethods disclosed herein and a pharmaceutically acceptable carrier ordiluent; and methods for the treatment of inflammation, septic shock,cancer or endocrine disorders and gastrointestinal disorders therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing in vitro biostability of somatostatin andthree analogs thereof in human serum. The graph depicts the percentageof undegraded molecules for each of the compounds initially and aftervarious periods of time.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All abbreviations used are in accordance with the IUPAC-IUBrecommendations on Biochemical Nomenclature (J. Biol. Chem.,247:977-983, 1972) and later supplements.

As used herein and in the claims, the phrase “an amino acid side chain”refers to the distinguishing substituent attached to the α-carbon of anamino acid; such distinguishing groups are well known to those skilledin the art. For instance, for the amino acid glycine, the R group is H;for the amino acid alanine, R is CH₃, and so on. Other typical sidechains of amino acids include the groups: (CH₃)₂CH—, (CH₃)₂CHCH₂—,CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—,NH₂C(═O)(CH₂)₂—, NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—,C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, andmethylimidazole.

As used herein and in the claims, the letters “(AA)” and the term “aminoacid” are intended to include common natural or synthetic amino acids,and common derivatives thereof, known to those skilled in the art,including but not limited to the following. Typical amino-acid symbolsdenote the L configuration unless otherwise indicated by D appearingbefore the symbol.

Abbreviated Designation Amino Acids Abu α-Amino butyric acid AlaL-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic acid βAsp(Ind)β-Indolinyl aspartic acid Cys L-Cysteine Glu L-Glutamic acid GlnL-Glutamine Gly Glycine His L-Histidine Hyp trans-4-L-Hydroxy ProlineIle L-Isoleucine Leu L-Leucine Lys L-Lysine Met L-Methionine Nalβ-Naphthyl alanine Orn Ornithine Phe L-Phenylalanine Pro L-Proline SerL-Serine Thr L-Threonine Trp L-Tryptophane Tyr L-Tyrosine Val L-Valine

Typical protecting groups, coupling agents, reagents and solvents suchas but not limited to those listed below have the followingabbreviations as used herein and in the claims. One skill in the artwould understand that the compounds listed within each group may be usedinterchangeably; for instance, a compound listed under “reagents andsolvents” may be used as a protecting group, and so on. Further, oneskill in the art would know other possible protecting groups, couplingagents and reagents/solvents; these are intended to be within the scopeof this invention.

Abbreviated Designation Protecting Groups Ada Adamantane acetyl AllocAllyloxycarbonyl Allyl Allyl ester Boc tert-butyloxycarbonyl Bzl BenzylFmoc Fluorenylmethyloxycarbonyl OBzl Benzyl ester OEt Ethyl ester OMeMethyl ester Tos (Tosyl) p-Toluenesulfonyl Trt Triphenylmethyl ZBenzyloxycarbonyl Abbreviated Designation Coupling Agents BOPBenzotriazol-1-yloxytris- (dimethyl-amino) phosphoniumhexafluorophosphate DIC Diisopropylcarbodiimide HBTU2-(1H-Benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphatePyBrOP Bromotripyrrolidinophosphonium hexafluorophosphate PyBOPBenzotriazol-1-yl-oxy-tris- pyrrolidino-phosphonium hexafluorophosphateTBTU O-(1,2-dihydro-2-oxo-1-pyridyl)- N,N,N′,N′-tetramethyluroniumtetrafluoroborate Reagents Abbreviated Designation and Solvents ACNAcetonitrile AcOH Acetic acid Ac₂O Acetic acid anhydride AdacOHAdamantane acetic acid Alloc-Cl Allyloxycarbonyl chloride Boc₂O Di-tertbutyl dicarbonate DMA Dimethylacetamide DMF N,N-dimethylformamide DIEADiisopropylethylamine Et₃N Triethylamine EtOAc Ethyl acetate FmocOSu9-fluorenylmethyloxy carbonyl N-hydroxysuccinimide ester HOBT1-Hydroxybenzotriazole HF Hydrofluoric acid MeOH Methanol Mes (Mesyl)Methanesulfonyl NMP 1-methyl-2-pyrrolidinone nin. Ninhydrin i-PrOHIso-propanol Pip Piperidine PP 4-pyrrolidinopyridine Pyr Pyridine SRIFSomatotropin release inhibiting factor SST Somatostatin SSTRSomatostatin receptor TEA Triethylamine TFA Trifluoroacetic acid THFTetrahydrofuran Triflate (Trf) Trifluoromethanesulfonyl Trf₂OTrifluoromethanesulfonic acid anhydride

The compounds herein described may have asymmetric centers. All chiral,diastereomeric, and racemic forms are included in the present invention.Many geometric isomers of olefins and the like can also be present inthe compounds described herein, and all such stable isomers arecontemplated in the present invention.

By “stable compound” or “stable structure” is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and Formulation into an efficacioustherapeutic agent.

As used herein and in the claims, “alkyl” or “alkylenyl” is intended toinclude both branched and straight-chain saturated aliphatic hydrocarbongroups having one to ten carbon atoms; “alkenyl” is intended to includehydrocarbon chains of either a straight or branched configuration andone or more unsaturated carbon-carbon bonds which may occur in anystable point along the chain, such as ethenyl, propenyl, and the like;and “alkynyl” is intended to include hydrocarbon chains of either astraight or branched configuration and one or more triple carbon-carbonbonds which may occur in any stable point along the chain, such asethynyl, propynyl, and the like.

As used herein and in the claims, “aryl” is intended to mean any stable5- to 7-membered monocyclic or bicyclic or 7- to 14-membered bicyclic ortricyclic carbon ring, any of which may be saturated, partiallyunsaturated or aromatic, for example, phenyl, naphthyl, indanyl, ortetrahydronaphthyl tetralin, etc.

As used herein and in the claims, “alkyl halide” is intended to includeboth branched and straight-chain saturated aliphatic hydrocarbon groupshaving one to ten carbon atoms, wherein 1 to 3 hydrogen atoms have beenreplaced by a halogen atom such as Cl, F, Br, and I.

As used herein and in the claims, the term “heterocyclic” is intended tomean any stable 5- to 7-membered monocyclic or bicyclic or 7- to10-membered bicyclic heterocyclic ring, which is either saturated orunsaturated, and which consists of carbon atoms and from 1 to 3heteroatoms selected from the group consisting of N, O and S and whereinthe nitrogen and sulfur atoms may optionally be oxidized, and thenitrogen atom optionally be quaternized, and including any bicyclicgroup in which any of the above-defined heterocyclic rings is fused to abenzene ring. The heterocyclic ring may be attached to its pendant groupat any heteroatom or carbon atom which results in a stable structure.The heterocyclic rings described herein may be substituted on carbon oron a nitrogen atom if the resulting compound is stable. Examples of suchheterocycles include, but are not limited to pyridyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl,piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oroctahydroisoquinolinyl and the like.

As used herein and in the claims, the phrase “therapeutically effectiveamount” means that amount of novel backbone cyclized peptide analog orcomposition comprising same to administer to a host to achieve thedesired results for the indications described herein, such as but notlimited of inflammation, septic shock, cancer, endocrine disorders andgastrointestinal disorders.

The term, “substituted” as used herein and in the claims, means that anyone or more hydrogen atoms on the designated atom is replaced with aselection from the indicated group, provided that the designated atom'snormal valency is not exceeded, and that the substitution results in astable compound.

When any variable (for example R, x, z, etc.) occurs more than one timein any constituent or in Formulae (I to XX) or any other Formula herein,its definition on each occurrence is independent of its definition atevery other occurrence. Also, combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds.

Synthetic Approach

According to the present invention peptide analogs are cyclized viabridging groups attached to the alpha nitrogens of amino acids thatpermit novel non-peptidic linkages. In general, the procedures utilizedto construct such peptide analogs from their building units rely on theknown principles of peptide synthesis; most conveniently, the procedurescan be performed according to the known principles of solid phasepeptide synthesis. The innovation requires replacement of one or more ofthe amino acids in a peptide sequence by novel building units of thegeneral Formula:

wherein R is the side chain of an amino acid, X is a spacer group and Gis the functional end group by means of which cyclization will beeffected. The side chain R is the side chain of any natural or syntheticamino acid that is selected to be incorporated into the peptide sequenceof choice. X is a spacer group that is selected to provide a greater orlesser degree of flexibility in order to achieve the appropriateconformational constraints of the peptide analog. Such spacer groupsinclude alkylene chains, substituted, branched and unsaturatedalkylenes, arylenes, cycloalkylenes, unsaturated and substitutedcycloakylenes. Furthermore, X and R can be combined to form aheterocyclic structure.

A preferred embodiment of the present invention utilizes alkylene chainscontaining from two to ten carbon atoms.

The terminal (ω) functional groups to be used for cyclization of thepeptide analog include but are not limited to:

a. Amines, for reaction with electrophiles such as activated carboxylgroups, aldehydes and ketones (with or without subsequent reduction),and alkyl or substituted alkyl halides.

b. Alcohols, for reaction with electrophiles such as activated carboxylgroups.

c. Thiols, for the formation of disulfide bonds and reaction withelectrophiles such as activated carboxyl groups, and alkyl orsubstituted alkyl halides.

d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.

e. Alkynes or Substituted Alkynes, for reaction with nucleophiles suchas amines, thiols or carbanions; free radicals; electrophiles such asaldehydes and ketones, and alkyl or substituted alkyl halides; ororganometallic complexes.

f. Carboxylic Acids and Esters, for reaction with nucleophiles (with orwithout prior activation), such as amines, alcohols, and thiols.

g. Alkyl or Substituted Alkyl Halides or Esters, for reaction withnucleophiles such as amines, alcohols, thiols, and carbanions (fromactive methylene groups such as acetoacetates or malonates); andformation of free radicals for subsequent reaction with alkenes orsubstituted alkenes, and alkynes or substituted alkynes.

h. Alkyl or Aryl Aldehydes and Ketones for reaction with nucleophilessuch as amines (with or without subsequent reduction), carbanions (fromactive methylene groups such as acetoacetates or malonates), diols (forthe formation of acetals and ketals).

i. Alkenes or Substituted Alkenes, for reaction with nucleophiles suchas amines, thiols, carbanions, free radicals, or organometalliccomplexes.

j. Active Methylene Groups, such as malonate esters, acetoacetateesters, and others for reaction with electrophiles such as aldehydes andketones, alkyl or substituted alkyl halides.

It will be appreciated that during synthesis of the peptide thesereactive end groups, as well as any reactive side chains, must beprotected by suitable protecting groups. Suitable protecting groups foramines are alkyloxy, substituted alkyloxy, and aryloxy carbonylsincluding, but not limited to, tert butyloxycarbonyl (Boc),Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) andBenzyloxycarbonyl (Z).

Carboxylic end groups for cyclizations may be protected as their alkylor substituted alkyl esters or thio esters or aryl or substituted arylesters or thio esters. Examples include but are not limited to tertiarybutyl ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethyl esterand 9-methyl fluorenyl.

Thiol groups for cyclizations may be protected as their alkyl orsubstituted alkyl thio ethers or disulfides or aryl or substituted arylthio ethers or disulfides. Examples of such groups include but are notlimited to tertiary butyl, trityl(triphenylmethyl), benzyl,2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl,carboxy-methyl, 2-thio-4-nitropyridyl.

It will further be appreciated by the artisan that the various reactivemoieties will be protected by different protecting groups to allow theirselective removal. Thus, a particular amino acid will be coupled to itsneighbor in the peptide sequence when the N^(α) is protected by, forinstance, protecting group A. If an amine is to be used as an end groupfor cyclization in the reaction scheme the N^(ω) will be protected byprotecting group B, or an ε amino group of any lysine in the sequencewill be protected by protecting group C, and so on.

The coupling of the amino acids to one another is performed as a seriesof reactions as is known in the art of peptide synthesis. Novel buildingunits of the invention, namely the N^(α)-ω functionalized amino acidderivatives are incorporated into the peptide sequence to replace one ormore of the amino acids. If only one such N^(α)-ω functionalized aminoacid derivative is selected, it will be cyclized to a side chain ofanother amino acid in the sequence. For instance: (a) an N^(α)-(ω-aminoalkylene) amino acid can be linked to the carboxyl group of an asparticor glutamic acid residue; (b) an N^(α)-(ω-carboxylic alkylene) aminoacid can be linked to the ε-amino group of a lysine residue; (c) anN^(α)-(ω-thio alkylene) amino acid can be linked to the thiol group of acysteine residue; and so on. A more preferred embodiment of theinvention incorporates two such N^(α)-ω-functionalized amino acidderivatives which may be linked to one another to form N-backbone toN-backbone cyclic peptide analogs. Three or more such building units canbe incorporated into a peptide sequence to create bi-cyclic peptideanalogs as will be elaborated below. Thus, peptide analogs can beconstructed with two or more cyclizations, including N-backbone toN-backbone, as well as backbone to side-chain or any other peptidecyclization.

As stated above, the procedures utilized to construct peptide analogs ofthe present invention from novel building units generally rely on theknown principles of peptide synthesis. However, it will be appreciatedthat accommodation of the procedures to the bulkier building units ofthe present invention may be required. Coupling of the amino acids insolid phase peptide chemistry can be achieved by means of a couplingagent such as but not limited to dicyclohexycarbodiimide (DCC),bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP—Cl),benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate(BOP), 1-oxo-1-chlorophospholane (Cpt—Cl), hydroxybenzotriazole (HOBT),or mixtures thereof.

It has now been found that coupling of the bulky building units of thepresent invention may require the use of additional coupling reagentsincluding, but not limited to: coupling reagents such as PyBOP®(Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), PyBrOP® (Bromo-tris-pyrrolidino-phosphoniumhexafluoro-phosphate), HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate), TBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate).

Novel coupling chemistries may be used, such as pre-formedurethane-protected N-carboxy anhydrides (UNCA's) and pre-formed acylfluorides. Said coupling may take place at room temperature and also atelevated temperatures, in solvents such as toluene, DCM(dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP(N-methyl pyrrolidinone) or mixtures of the above.

One object of the present invention is a method for the preparation ofbackbone cyclized peptide analogs of Formula (I):

wherein the substituents are as defined above;

comprising the steps of incorporating at least oneN^(α)-ω-functionalized derivatives of amino acids of Formula (VI):

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R′ is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; B is a protectinggroup selected from the group consisting of alkyloxy, substitutedalkyloxy, or aryloxy carbonyls; and G is a functional group selectedfrom the group consisting of amines, thiols, alcohols, carboxylic acidsand esters, aldehydes, alcohols and alkyl halides; and A is a specificprotecting group of G;

with a compound of the Formula (VII):

H₂N—(AA)_(f)—CO—E  Formula (VII)

wherein f is an integer from 1 to 10; (AA) designates an amino acidresidue wherein the amino acid residues may be the same or different,and E is a hydroxyl, a carboxyl protecting group or an amide to give acompound of the general Formula:

(ii) selectively removing protecting group B and reacting theunprotected compound with a compound of Formula:

B—NH—(AA)_(e)—COOH  Formula (IX)

wherein B and (AA) are as described above and e is an integer from 1 to10,

to give a compound of Formula:

wherein B, (AA), e, R¹, and f are as described above;

(iii) removing the protecting group B from the compound of the Formula(X) and reacting the unprotected compound with a compound of Formula:

wherein X′ is a spacer group selected from the group consisting ofalkylenes, substituted alkylenes, arylenes, cycloalkylenes andsubstituted alkylenes; G′ is a functional group selected from amines,thiols, carboxyls, aldehydes or alcohols; A′ is a specific-protectinggroup thereof; R¹ is an amino acid side chain such as H, CH₃, etc.,optionally bound with a specific protecting group; and B is a protectinggroup;

to yield a compound of Formula:

(iv) removing the protecting group B and reacting the unprotectedcompound with a compound of Formula:

B—NH—(AA)_(d)—COOH  Formula (IXa)

to yield a compound of Formula:

(v) selectively removing protecting groups A and A′ and reacting theterminal groups G and G′ to form a compound of the Formula:

wherein d, e and f are independently an integer from 1 to 10; (AA) is anamino acid residue wherein the amino acid residues in each chain may bethe same or different; E is an hydroxyl group, a carboxyl protectinggroup or an amino group; R and R′ are independently an amino acidside-chain such as H, CH₃, etc.; and the line designates a bridginggroup of the Formula: —X—M—Y—W—Z—

wherein M and W are independently selected from the group consisting ofdisulfide, amide, thioether, imine, ether, and alkene; X, Y and Z areindependently selected from the group consisting of alkylene,substituted alkylene, arylene, cycloalkylene, and substitutedcycloalkylene;

(vi) removing all remaining protecting groups to yield a compound ofFormula (I).

Bicyclic analogs are prepared in the same manner, that is, by repetitionof steps (v) and (vi). The determination of which residues are cyclizedwith which other residues is made through the choice of blocking groups.The various blocking groups may be removed selectively, thereby exposingthe selected reactive groups for cyclization.

Preferred are methods for the preparation of backbone cyclized peptideanalogs of Formula (I) wherein G is an amine, thiol or carboxyl group; Rand R¹ are each other than H, such as CH₃, (CH₃)₂CH—, (CH₃)₂CHCH₂—,CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—,NH₂C(═O) (CH₂)₂—, HOC(═O)CH₂—, HOC(═O) (CH₂)₂—, NH₂(CH₂)₄—, C(NH₂)₂NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl, methylindole, and methylimidazole,and wherein E is covalently bound to an insoluble polymeric support.

Another object of the present invention is a method for the preparationof backbone cyclized peptide analogs of Formula (II):

wherein the substituents are as defined above;

comprising the steps of: incorporating at least one ω-functionalizedamino acid derivative of the general Formula (VI):

wherein X is a spacer group selected from the group consisting ofalkylene, substituted alkylene, arylene, cycloalkylene and substitutedcycloalkylene; R is the side chain of an amino acid, such as H, CH₃,etc.; B is a protecting group selected from the group consisting ofalkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is afunctional group selected from the group consisting of amines, thiols,alcohols, carboxylic acids and esters or alkyl halides and A is aprotecting group thereof;

into a peptide sequence and subsequently selectively cyclizing thefunctional group with one of the side chains of the amino acids in saidpeptide sequence.

Preferred is the method for the preparation of backbone cyclized peptideanalogs of Formula (II) wherein G is a carboxyl group or a thiol group;R is CH₃, (CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—,CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—, HOC(═O)CH₂—,HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—, benzyl,methylindole, and methylimidazole, and wherein E is covalently bound toan insoluble polymeric support.

Preparation of backbone to side chain cyclized peptide analogs isexemplified in Scheme I below. In this schematic example, the bridginggroup consists of alkylene spacers and an amide bond formed between anacidic amino acid side chain (e.g. aspartic or glutamic acid) and anω-functionalized amino acid having a terminal amine.

(Scheme I) Preparation of Peptides with Backbone to Side ChainCyclization

One preferred procedure for preparing the desired backbone cyclicpeptides involves the stepwise synthesis of the linear peptides on asolid support and the backbone cyclization of the peptide either on thesolid support or after removal from the support. The C-terminal aminoacid is bound covalently to an insoluble polymeric support by acarboxylic acid ester or other linkages such as amides. An example ofsuch support is a polystyrene-co-divinyl benzene resin. The polymericsupports used are those compatible with such chemistries as Fmoc and Bocand include for example PAM resin, HMP resin and chloromethylated resin.The resin bound amino acid is deprotected for example with TFA to give(1) below and to it is coupled the second amino acid, protected on theN^(α) for example by Fmoc, using a coupling reagent like BOP. The secondamino acid is deprotected to give (3) using for example piperidine 20%in DMF. The subsequent protected amino acids can then be coupled anddeprotected at ambient temperature. After several cycles of coupling anddeprotection that gives peptide (4), an amino acid having for examplecarboxy side chain is coupled to the desired peptide. One such aminoacid is Fmoc-aspartic acid t-butyl ester. After deprotection of theN^(α) Fmoc protecting group that gives peptide (5), the peptide is againelongated by methods well known in the art to give (6). Afterdeprotection a building unit for backbone cyclization (the preparationof which is described in Schemes III-VIII) is coupled to the peptideresin using for example the coupling reagent BOP to give (7). One suchbuilding unit is for example Fmoc-N^(α)(ω-Boc-amino alkylene)amino acid.After deprotection the peptide can then be elongated, to the desiredlength using methods well known in the art to give (8). The coupling ofthe protected amino acid subsequent to the building unit is performed bysuch coupling agents exemplified by PyBrOP® to ensure high yield.

After the linear, resin bound peptide, e.g. (8), has been prepared theω-alkylene-protecting groups for example Boc and t-Bu are removed bymild acid such as TFA to give (9). The resin bound peptide is thendivided into several parts. One part is subjected to on-resincyclization using for example TBTU as cyclization agent in DMF to ensurehigh yield of cyclization, to give the N-backbone to side chain cyclicpeptide resin (10). After cyclization on the resin the terminal aminoprotecting group is removed by agents such as piperidine and thebackbone to side chain cyclic peptide (11) is obtained after treatmentwith strong acid such as HF. Alternatively, prior to the removal of thebackbone cyclic peptide from the resin, the terminal amino group isblocked by acylation with agents such as acetic anhydride, benzoicanhydride or any other acid such as adamantyl carboxylic acid activatedby coupling agents such as BOP.

The other part of the peptide-resin (9) undergoes protecting of the sidechains used for cyclization, for example the ω-amino and carboxy groups.This is done by reacting the ω-amino group with for example Ac₂O andDMAP in DMF and activating the free ω-carboxy group by for example DICand HOBT to give the active ester which is then reacted with for exampleDh₃NH₂ to give the linear analog (13) of the cyclic peptide (10).Removal of the peptide from the resin and subsequent removal of the sidechains protecting groups by strong acid such as HF to gives (14) whichis the linear analog of the backbone to side chain cyclic peptide (11).

The linear analogs are used as reference compounds for the biologicalactivity of their corresponding cyclic compounds.

[Reaction Scheme I follows at this point]

The selection of N^(α) and side chain protecting groups is, in part,dictated by the cyclization reaction which is done on the peptide-resinand by the procedure of removal of the peptide from the resin. The N^(α)protecting groups are chosen in such a manner that their removal willnot effect the removal of the protecting groups of theN^(α)(ω-aminoalkylene) protecting groups. In addition, the removal ofthe N^(α)(ω-aminoalkylene)protecting groups or any other protectinggroups on ω-functional groups prior to the cyclization, will not effectthe other side chain protection and/or the removal of the peptide fromthe resin. The selection of the side chain protecting groups other thanthose used for cyclization is chosen in such a manner that they can beremoved subsequently with the removal of the peptide from the resin.Protecting groups ordinarily employed include those which are well knownin the art, for example, urethane protecting substituents such as Fmoc,Boc, Alloc, Z and the like.

It is preferred to utilize Fmoc for protecting the α-amino group of theamino acid undergoing the coupling reaction at the carboxyl end of saidamino acid. The Fmoc protecting group is readily removed following suchcoupling reaction and prior to the subsequent step by the mild action ofbase such as piperidine in DMF. It is preferred to utilize Boc forprotecting ω-amino group of the N^(α)(ω-aminoalkylene) group and t-Bufor protecting the carboxy group of the amino acids undergoing thereaction of backbone cyclization. The Boc and t-Bu protecting groups arereadily removed simultaneously prior to the cyclization.

(Scheme II) Preparation of Peptides with Backbone to BackboneCyclization

Preparation of N-backbone to N-backbone cyclized peptide analogs isexemplified in scheme II. In this schematic example, the building groupconsists of alkylene spacers and two amide bonds.

A building unit for backbone cyclization (the preparation of whichdescribed in Schemes III-VIII) is coupled to a peptide resin, forexample peptide-resin (4), using for example the coupling reagent BOP togive (16). One such building unit is for example Fmoc-Nα(ω-Boc-aminoalkylene)amino acid. The side chain Boc protecting group is removed bymild acid such as TFA in DCM and an N-Boc protected ω-amino acid, or anyother Boc protected amino acid, is coupled to the side chain amino groupusing coupling agent such as BOP to give peptide-resin (17).

After deprotection of the N^(α) Fmoc protecting group by mild base suchas piperidine in DMF, the peptide can then be elongated, if required, tothe desired length using methods well known in the art to give (18).Alternatively, the deprotection of the N^(α) Fmoc and subsequentelongation of the peptide can be done before deprotection of the sidechain Boc protecting group. The elongation of the N-alkylene side chainallow control of the ring size. The coupling of the protected amino acidsubsequent to the building unit is performed by such coupling agentsexemplified by PyBrOP® to ensure high yield.

After deprotection of the terminal N^(α) Fmoc group, a second buildingunit, for example Fmoc-N^(α)(ω-t-Bu-carboxyalkylene)amino acid iscoupled to the peptide-resin to give (19). After deprotection of theN^(α) Fmoc protecting group, the peptide can then be elongated, ifrequired, to the desired length using methods well known in the art togive (20). The coupling of the protected amino acid subsequent to thebuilding unit is performed by such coupling agents exemplified byPyBrOP® to ensure high yield. After the linear, resin bound peptide,e.g. (20), has been prepared the ω-alkylene-protecting groups forexample Boc and t-Bu are removed by mild acid such as TFA to give (21).The resin peptide is then divided into several parts. One part issubjected to on-resin cyclization using for example TBTU as cyclizationagent in DMF to ensure high yield of cyclization, to give the N-backboneto N-backbone cyclic peptide resin (22). After cyclization on the resinthe terminal amino protecting group is removed by agents such aspiperidine and the backbone to backbone cyclic peptide (23) is obtainedafter treatment with strong acid such as HF. Alternatively, prior to theremoval of the backbone cyclic peptide from the resin, the terminalamino group of (22) is blocked, after deprotection, by acylation withagents such as acetic anhydride, benzoic anhydride or any other acidsuch as adamantyl carboxylic acid activated by coupling agents such asBOP to give the N-terminal blocked backbone to backbone cyclic peptide(24).

The other part of the peptide-resin (21) undergoes protecting of theside chains used for cyclization, for example the ω-amino and carboxygroups. This is done by reacting the ω-amino group with for example Ac₂Oand DMAP in DMF and activating the free ω-carboxy group by for exampleDIC and HOBT to give the active ester which is then reacted with forexample MeNH₂. Removal of the peptide from the resin and subsequentremoval of the side chains protecting groups by strong acid such as HFto gives (26) which is the linear analog of the backbone to backbonecyclic peptide (23). The linear analogs are used as reference compoundsfor the biological activity of their corresponding cyclic compounds.

Reaction Scheme II Follows at this Point

Novel Synthesis of Building Units

The novel synthesis providing N(ω-(functionalized) alkylene) amino acidsused to generate backbone cyclic peptides is depicted in schemesIII-VIII. In this approach we have implemented the following changes inorder to devise a practical, general synthesis:

1. The nucleophile is a secondary nitrogen, which is a betternucleophile than the primary nitrogen previously used. This alsoprevents the possibility of double alkylation.

2. The leaving group was changed to trifluoromethanesulfonyl (triflate),which has a much lower tendency to eliminate than a halogen, thus makingit possible to implement the synthesis with amino acids other thanglycine. Furthermore, the triflate leaving group prevents racemizationduring the alkylation reaction.

3. The carboxylate is esterified prior to the substitution reaction, tofacilitate the substitution by removing the negative charge next to theelectrophilic carbon.

(Scheme III) Preparation of N^(α), N^(ω) Protected ω-amino AlkyleneAmino Acids Building Units

One preferred procedure for the preparation of protected N^(α)(ω-aminoalkylene) amino acids involves the N^(α) alkylation of suitablyprotected diamino alkanes. One preferred N^(α),N^(ω) di- protecteddiamino alkane is for example N^(α)-Benzyl, N^(ω)-Boc diamino alkane(27). This starting material contains one protecting group such as Bocwhich is necessary for the final product, and a temporary protectinggroup such as Bzl to minimize unwanted side reactions during thepreparation of the titled compound. One preferred procedure for thepreparation of the starting material (27) involves reductive alkylationof N-Boc diamino alkane with aldehydes such as benzaldehyde. Thetemporary protection of the N^(α) amino group, which is alkylated in thereaction by such protecting groups as Bzl, minimizes the dialkylationside reaction and allows removal by such conditions that do not removethe N^(ω)-protecting group.

The N^(α),N^(ω) di-protected diamino alkane is reacted with for examplechiral α-hydroxy α-substituted acid esters where the hydroxyl moiety isconverted to a leaving group for example Triflate.

The use of Triflate as the leaving group was found to be superior toother leaving groups such as halogens, Tosyl, Mesyl, etc., because itprevents the β-elimination reaction encountered with the other leavinggroups. The use of Triflate as the leaving group also ensures highoptical purity of the product (28). The temporary N^(α) protectinggroup, such as Bzl, and the carboxyl protecting group, such as methylester, are removed by mild conditions, such as catalytic hydrogenationand hydrolysis, that do not remove the N^(ω) protecting group such asBoc to give the N^(ω) protected amino acid (29). Introduction of theN^(α) protecting group suitable for peptide synthesis is accomplished bymethods well known in the art, to give the protected N^(α)(N^(ω)protected amino alkylene) amino acid (30).

The choice of the N^(α) and the N^(ω) protecting groups is dictated bythe use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. Combinations of N^(α) andN^(ω) protecting groups are for example: N^(α)-Fmoc, N^(ω)-Boc;N^(α)-Fmoc, N^(ω)-Alloc; N^(α)-Boc, N^(ω)-Alloc. These combinations aresuitable for peptide synthesis and backbone cyclization, either on solidsupport or in solution.

(Scheme IV) Preparation of N^(α), N^(ω) Protected ω-amino AlkyleneGlycine Building Units

One preferred procedure for the preparation of protected N^(α)(ω-aminoalkylene) glycines involves the reaction of the N^(α), N^(ω)di-protected di amino alkane (27) with commercially availableα-activated carboxylic acid esters, for example benzylbromo acetate.Since the titled compound is achiral, the use of leaving groups such asTrf, Tos or Mes is not necessary. The use of the same temporaryprotecting groups for the N^(α) and the carboxy groups, for example theBzl protecting group, ensures the prevention of the undesireddialkylation side reaction and allows concomitant removal of thetemporary protecting groups thus giving high yield of the N^(ω)protected amino acid (32). Introduction of the N^(α) protecting groupsuitable for peptide synthesis is accomplished by methods well known inthe art, to give the protected N^(α) (N^(ω) protected amino alkylene)glycines (33).

The choice of the N^(α) and the N^(ω) protecting groups is dictated bythe use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. Combinations of N^(α) andN^(ω) protecting groups are for example: N^(α) Fmoc, N^(ω) Boc ; N^(α)Fmoc, N^(ω) Alloc; N^(α) Boc, N^(ω) Alloc. These combinations aresuitable for peptide synthesis and backbone cyclization, either on solidsupport or in solution.

(Scheme V) Preparation of N^(α), ω-carboxy Protected ω-carboxy AlkyleneAmino Acids

One preferred procedure for the preparation of protected N^(α)(ω-carboxyalkylene) amino acids involves the N^(α)-alkylation of suitably N^(α),ω-carboxy deprotected amino acids. One preferred deprotected amino acidis N^(α)-Benzyl ω-amino acids t-butyl esters (34). This startingmaterial contains one protecting group such as t-Bu ester which isnecessary for the final product, and a temporary protecting group suchas N^(α) Bzl to minimize side reactions during the preparation of thetitled compound. One preferred procedure for the preparation of thestarting material (34) involves reductive alkylation of ω-amino acidst-butyl esters with aldehydes such as benzaldehyde. The temporaryprotection of the amino group which is used as nucleophile in theproceeding alkylation reaction by such protecting groups as Bzlminimizes the dialkylation side reaction.

The N^(α), ω-carboxy deprotected amino acids (34) are reacted with, forexample, chiral α-hydroxy α-substituted acid esters where the hydroxylmoiety is converted to a leaving group, for example, Triflate. The useof Triflate as the leaving group was found to be superior to otherleaving groups such as halogens, Tosyl, Mesyl; etc., because it preventsthe β-elimination reaction encountered with the other leaving groups.The use of Triflate as the leaving group also ensures high opticalpurity of the product, for example (36). The temporary N^(α) protectinggroup, such as Bzl, and the α-carboxyl protecting group, such as benzylester, are concomitantly removed by mild condition, such as catalytichydrogenation, that to not remove the ω-carboxy protecting group such ast-Bu to give the N^(α) (protected ω-carboxy alkylene) amino acid (36).Introduction of the N^(α) protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N^(α)(ω protected carboxy alkylene) amino acid (37).

The choice of the N^(α) and the ω-carboxy protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N^(α) andω-carboxy protecting groups are for example: N^(α)-Fmoc, ω-carboxy t-Bu;N^(α)-Fmoc, ω-carboxy Alloc; N^(α)-Boc, ω-carboxy Alloc. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

(Scheme VI) Preparation of N^(α), ω-carboxy Protected ω-carboxy AlkyleneGlycine Building Units

One preferred procedure for the preparation of protected N^(α)(ω-carboxyalkylene)glycines involves the N^(α)-alkylation of suitably N^(α),ω-carboxy deprotected amino acids (34) with commercially availableα-activated carboxylic acid esters for example, benzyl bromo acetate.Since the titled compound is achiral, the use of leaving groups such asTrf, Tos or Mes is not necessary.

The use of the same temporary protecting groups for the N^(α) and theα-carboxy groups, for example the Bzl protecting group, ensures theprevention of the undesired dialkylation side reaction and allowsconcomitant removal of the temporary protecting groups thus giving highyield of the N^(α)(protected ω-carboxy alkylene) glycines (39).Introduction of the N^(α) protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N^(α)(ω protected carboxy alkylene) glycines (40).

The choice of the N^(α) and the ω-carboxy protecting groups is dictatedby the use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N^(α) andω-carboxy protecting groups are, for example: N^(α) Fmoc, ω-carboxyt-Bu; N^(α) Fmoc, ω-carboxy Alloc; N^(α) Boc, ω-carboxy Alloc. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

(Scheme VII) Preparation of N^(α) S^(ω) Protected ω-thio Alkylene AminoAcid Building Units

One preferred procedure for the preparation of N^(α), S^(ω)-deprotectedN^(α)(ω-thio alkylene) amino acids involves the N^(α)-alkylation ofsuitably S^(ω) protected ω-thio amino alkanes. Suitable S^(ω) protectinggroups are, for example, Bzl, t-Bu, Trt. One preferred S^(ω)-protectedω-thio amino alkanes is for example ω-(S-Benzyl) amino alkanes (41). Onepreferred procedure for the preparation of the starting material (41)involves the use of salts of S-protected thiols as nucleophiles for anucleophilic substitution reaction on suitably N^(α)-protectedω-activated amino alkanes. Removal of the amino protection gives thestarting material (41).

The S-protected ω-thio amino alkanes (41) are reacted with for examplechiral α-hydroxy α-substituted acid esters where the hydroxyl moiety isconverted to a leaving group for example Triflate. The use of Triflateas the leaving group was found to be superior to other leaving groupssuch as halogens, Tosyl, Mesyl etc. because it prevents theβ-elimination reaction encountered with the other leaving groups. Theuse of Triflate as the leaving group also ensures high optical purity ofthe product for example (42). The temporary α-carboxyl protecting group,such as methyl ester, is removed by mild condition, such as hydrolysiswith base, that to not remove the ω-thio protecting group such as S-Bzlto give the N^(α) (S-protected ω-thio alkylene) amino acid (43).Introduction of the N^(α) protecting group suitable for peptidesynthesis is accomplished by methods well known in the art, to give theprotected N,S protected N^(α) (ω-thio alkylene) amino acid (44).

The choice of the N^(α) and the ω-thio protecting groups is dictated bythe use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N^(α) andω-thio protecting groups are for example: N^(α) Fmoc, S^(ω) t-Bu; N^(α)Fmoc, S^(ω) Bzl; N^(α) Fmoc, S^(ω) Trt; N^(α) Boc, S^(ω) Bzl. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

(Scheme VIII) Preparation of N^(α), S^(ω) Protected ω-thio AlkyleneGlycine Building Units

One preferred procedure for the preparation of N^(α), S^(ω)-deprotectedN^(α)(ω-thio alkylene) amino acids involves the N^(α)-alkylation ofsuitably S^(ω) protected ω-thio amino alkanes (41) with commerciallyavailable α-activated carboxylic acid esters for example ethyl bromoacetate. Since the titled compound is achiral, the use of leaving groupssuch as Trf, Tos or Mes is not necessary.

Suitable protecting groups for the ω-thio groups are for example Bzl,t-Bu, Trt. One preferred S-protected ω-thio amino alkanes is for exampleω-(S-Benzyl) amino alkanes (41). The N-alkylation reaction gives theester (45). The temporary α-carboxyl protecting group, such as ethylester, is removed by mild conditions, such as hydrolysis with base, thatto not remove the ω-thio protecting group such as S-Bzl to give theN^(α) (S-protected ω-thio alkylene) glycines (46). Introduction of theN^(α) protecting group suitable for peptide synthesis is accomplished bymethods well known in the art, to give the protected N^(α),S^(ω)-deprotected N^(α) (ω-thio alkylene) glycines (47).

The choice of the N^(α) and the ω-thio protecting groups is dictated bythe use of the building units in peptide synthesis. The protectinggroups have to be orthogonal to each other and orthogonal to the otherside chains protecting groups in the peptide. A combination of N^(α) andω-thio protecting groups are for example: N^(α) Fmoc, S^(ω) t-Bu; N^(α)Fmoc, S^(ω) Bzl; N^(α) Fmoc, S^(ω) Trt; N^(α) Boc, S^(ω) Bzl. Thesecombinations are suitable for peptide synthesis and backbonecyclization, either on solid support or in solution.

SPECIFIC EXAMPLES OF PEPTIDES

Preparation of the novel backbone cyclized peptide analogs using theschematics outlined above will be illustrated by the followingnon-limiting specific examples:

EXAMPLE 1Ada-(D)Arg-Arg-cyclo(N^(α)(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OHSTAGE 1

Boc-Arg(Tos)-O-resin→Fmoc-Phe-Arg(Tos)-O-resin

Boc-L-Arg(Tos)-O-resin (0.256 g, 0.1 mmole, 0.39 meq of nitrogen/g) wasplaced in a shaker flask and swelled for two hours by the addition ofDCM. The resin was then carried out through the procedure in Table 1which includes two deprotections of the Boc protecting group with 55%TFA in DCM for a total of 22 minutes, washing, neutralization with 10%DIEA in NMP and washing (Table 1 steps 1-8). After positive ninhydrintest, as described in Kaiser et al., Anal Biochem., 34:595, 1970 and isincorporated herein by reference in its entirety, coupling (Table 1steps 9-10) was achieved in NMP by the addition of Fmoc-L-Phe (0.232 g,0.6 mmole) and after 5 minutes of shaking, solid BOP reagent (0.265 g,0.6 mmole) was added to the flask.

TABLE 1 PROCEDURE FOR 0.1 mMOLE SCALE VOL- RE- STEP SOLVENT/ UME TIMEPEAT NO. REAGENT (ML) (MIN) (XS) COMMENT 1 DCM 5 120  1 Swells resin 2DCM 5 2 3 3 TFA/DCM 55% 5 2 1 Deprotection 4 TFA/DCM 55% 5 20  1Deprotection 5 DCM 5 2 3 6 NMP 5 2 4 check for positive nin. 7 DIEA/NMP5 5 2 Neutralization 8 NMP 5 2 5 9 Fmoc-AA in NMP 5 5 Coupling add BOP 6eq. add DIEA 120 600 1 12 eq. Check pH, adjust to pH 8 with DIEA 10  NMP5 2 5 check for negative nin. 11  Pip/NMP 20% 5 10  1 Deprotection 12 Pip/NMP 20% 5 10  1 13  NMP 5 2 6 check for positive nin.

After shaking for 10 minutes, the mixture was adjusted to pH 8 (measuredwith wetted pH stick) by the addition of DIEA (0.209 mL, 1.2 mmole) andthe flask shaken for 10 hours at ambient temperature. The resin was thenwashed and subjected to ninhydrin test. After negative ninhydrin testthe resin was used for the next coupling.

STAGE 2

Fmoc-Phe-Arg(Tos)-O-resin→Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to twodeprotections of the Fmoc protecting group by 20% Pip in NMP (Table 1steps 11-13). After washing and ninhydrin test (Method J, below),coupling of Fmoc-D-Phe was achieved as described in Stage 1 (Table 1steps 9-10) using Fmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and theFmoc group deprotected as described above (Table 1 steps 11-13) Afterwashing and ninhydrin test, coupling of Fmoc-D-Asp(t-Bu) was achieved asdescribed in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Asp(t-Bu) (0.247g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed and the Fmoc group deprotected as describedabove (Table 1 steps 11-13). After washing and ninhydrin test, couplingof Fmoc-L-Phe was achieved as described in Stage 1 (Table 1 steps 9-10)using Fmoc-L-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1 steps 11-13). After washing andninhydrin test, coupling of Fmoc-L-Hyp(OBzl) was achieved as describedin Stage 1 (Table 1 steps 9-10) using Fmoc-L-Hyp(OBzl) (0.266 g, 0.6mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).The resin was washed and the Fmoc group deprotected as described above(Table 1 steps 11-13). The resin was washed and subjected to picric acidtest (Method K). Coupling of Fmoc-N^(α)(6-Boc amino hexylene) glycinewas achieved as described in Stage 1 (Table 1 steps 9-10) usingFmoc-N^(α)(6-Boc amino hexylene)glycine (0.3 g, 0.6 mmole), BOP reagent(0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was thenwashed and subjected to the picric acid test (Method K below). Afternegative test the resin was used for the next coupling.

STAGE 3

Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin→Fmoc-D-Arg(Tos)-Ara(Tos)-N^(α)(6-Bocamino hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin

The Fmoc-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage2) was subjected to three deprotection of the Fmoc protecting group by20% Pip in NMP (Table 2 steps 1-2). After washing, the picric acid test(Method K) was performed. If the test did not show 98±2%, deprotectionof the peptide resin was subjected again to 3 deprotection steps (Table2 steps 1-2), washing and picric acid test (Method K). Coupling ofFmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6mmole) and after 5 minutes of shaking, solid PYBOP reagent (0.28 g, 0.6mmole) was added to the flask. After shaking for 10 minutes, the mixturewas adjusted to pH 8 (measured with wetted pH stick) by the addition ofDIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambienttemperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Method K) (Table 2 steps 3-6). If thetest did not show 98±2% coupling the peptide resin was subjected againto a third coupling for 2 hours at 50° C. (Table 2 step 7). The resinwas washed subjected to three deprotection of the Fmoc protecting groupby 20% Pip in NMP (Table 2 steps 1-2). After washing picric acid test(Method K) was performed.

TABLE 2 PROCEDURE FOR 0.1 mMOLE SCALE VOL- RE- STEP SOLVENT/ UME TIMEPEAT NO. REAGENT (ML) (MIN) (XS) COMMENT 1 Piperidine/NMP 5 10  3Deprotection 20% 2 NMP 5 2 6 Picric acid test. 3 Fmoc-AA in NMP 5 5Coupling add PyBroP 6 eq. add DIEA 150  1 12 eq. Check pH, adjust to pH8 with DIEA. 4 NMP 5 2 3 check for negative 5 Fmoc-AA in NMP 5 5Coupling add PyBroP 6 eq. add DIEA 20 hr. 1 12 eq. Check pH, adjust topH 8 with DIEA. 6 NMP 5 2 4 Picric acid test. If less than 98 ± 2%coupling repeat Steps 4-5 7 Fmoc-AA in NMP 5 5 Coupling at add PyBOP 50°C. 6 eq. add DIEA 120  1 12 eq. Check pH, adjust to pH 8 with DIEA. 8NMP 5 2 4

If the test did not show 98±2% deprotection, the peptide resin wassubjected again to 3 deprotection steps (Table 2 steps 1-2), washing andthe picric acid test (Method K). Coupling of Fmoc-D-Arg(Tos) wasachieved in NMP as described in Stage 1 (Table 1 steps 9-10) usingFmoc-D-Arg(Tos) (0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole)and DIEA (0.209 mL, 1.2 mmole). The resin was washed 6 times with NMP(Table 1 step 15) and used in the next stages.

STAGE 4

Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin→Ada-D-Arg-Arg-cyclo(N^(α)(1-(6-amidohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH

The Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-Boc aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage3) was subjected to deprotection of the Boc and t-Bu protecting groupsand on resin cyclization according to Table 3. The peptide resin waswashed with DCM and deprotected as described in Stage 1 by 55% TFA inDCM. After washing and neutralization by 10% DIEA in NMP and washing 6times with DCM the peptide resin was dried in vacuo for 24 hours. Thedry peptide resin weight, 0.4 g, it was divided into two parts. 0.2 g ofthe peptide resin was swollen 2 hours in 5 mL NMP and cyclized asfollows: Solid TBTU reagent (0.19 g, 6 mmole) was added to the flask.After shaking for 10 minutes, the mixture was adjusted to pH 8 by theaddition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5hours at ambient temperature. The resin was then washed and subjected toa second coupling by the same procedure for 20 hours. After washing theresin was subjected to picric acid test (Method K) (Table 3 steps 8-11).If the test did not show 98±2% cyclization the peptide resin wassubjected again to a third cyclization for 2 hours at 50° C. (Table 2step 12). The resin was washed, subjected to three deprotection of theFmoc protecting group by 20% Pip in NMP (Table 2 steps 1-2). Afterwashing and ninhydrin test, the N-terminal amino group was blocked byAda. Adamantane acetic acid (0.108 g, 6 mmole), BOP reagent (0.265 g,0.6 mmole) and DIEA (0.209 mL, 1.2 mmole) were added and the flaskshaken for 2 hours. After washing 6 times with NMP (Table 2 step 13),ninhydrin test (Method J) was performed. If the test was positive orslightly positive the protecting with adamantane acetic acid wasrepeated. If the ninhydrin test was negative, the peptide resin waswashed 6 times with NMP and 6 times with DCM. The resin was dried undervacuum for 24 hours. The dried resin was subjected to HF as follows: tothe dry peptide resin (0.2 g) in the HF reaction flask, anisole (2 mL)was added and the peptide treated with 20 mL liquid HF at −20° C. for 2hours. After the evaporation of the HF under vacuum, the anisole waswashed with ether (20 mL, 5 times) and the solid residue dried invacuum. The peptide was extracted from the resin with TFA (10 mL, 3times) and the TFA evaporated under vacuum. The residue was dissolved in20 mL 30% AcOH and lyophilized. This process was repeated 3 times. Thecrude peptide was purified by semiprep HPLC (Method H). The finalproduct was obtained as white powder by lyophilization from dioxane,which gave 42 mg (56%) of the title compound.

HPLC (Method G) RT 32.15 minutes, 95%

TOF MS: 1351.4 (M⁺)

AAA in agreement with the title compound

TABLE 3 PROCEDURE FOR 0.05 mMOLE SCALE VOL- RE- STEP SOLVENT/ UME TIMEPEAT NO. REAGENT (ML) (MIN) (XS) COMMENT  1 DCM 5 2 3  2 TFA/DCM 55% 5 21 Deprotection  3 TFA/DCM 55% 5 20  1 Deprotection  4 DCM 5 2 3  5 NMP 52 4  6 DIEA/NMP 10% 5 5 2 Neutralization  7 NMP 5 2 5  8 TBTU/NMP/ 5150  3 Cyclization DIEA  9 NMP 5 2 4 Picric acid test. If less than 98 ±2% coupling perform Steps 10-12. If above 98 ± 2%, go to step 13. 10TBTU/NNP/ 5 20 hr 3 Cyclization. Check DIEA pH, adjust to pH 8 withDIEA. 11 NMP 5 2 4 Picric acid test. If less than 98 ± 2% couplingperform Steps 12. If above 98 ± 2%, go to step 13 12 TBTU/NMP/ 5 120  3Cyclization, 50 C. DIEA Check pH, adjust to pH 8 with DIEA. 13 NMP 5 2 614 Pip/NMP 20% 5 10  1 Deprotection 15 Pip/NMP 20% 5 10  1 16 NMP 5 2 6Check for positive nin. 17 AdacOH/BOP/ 5 2 1 NMP 18 NMP 5 2 6 Check fornegative nin. 19 DCM 5 2 4

EXAMPLE 2 NON-CYCLIZED PEPTIDE (Control for biological assays)

Ada-D-Arg-Arg-N^(α)(6-acetamidohexylene)Gly-Hyp-Phe-D-Asp(NH-Me)-D-Phe-Phe-Arg-OH

The Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(6-aminohexylene)Gly-Hyp(OBzl)-Phe-D-Asp-D-Phe-Phe-Arg(Tos)-O-resin (0.2 g)which was prepared in Example 1 Stage 4 was subjected to acetylation ofthe 6-amino side chain of N^(α)(6-acetamidohexylene)Gly and to methylamidation of the carboxylic group of D-Asp as described in Table 4. Thepeptide resin was swollen in 5 mL NMP for 2 hours and AcO (0.113 mL, 12mmole) and PP (17 mg) were added. After 30 minutes, the resin was washedwith NMP 6 times and subjected to ninhydrin test. If the test waspositive or slightly positive the acetylation reaction was repeated. Ifthe ninhydrin test was negative, the carboxy group of D-Asp wasactivated by the addition of HOBT (0.040 g, 0.3 mmole) and DIC (0.047mL, 0.3 mmole) to the peptide resin in NMP. The mixture was shaken forhalf an hour and a solution of 30% methylamine in EtOH (0.2 mL) wasadded. After one hour, the resin was washed 6 times with NMP and theterminal Fmoc group removed by 20% Pip in NMP (Table 4 steps 7-9). Afterwashing with NMP the N-terminal amino group was blocked by Ada asdescribed in Example 1 Stage 4 and the resin was washed with NMP and DCM(Table 4 steps 10-12) and the resin dried in vacuo. The peptide wasdeprotected and cleaved from the resin by HF. To the dry peptide resin(0.2 g) in the HF reaction flask, anisole (2 mL) was added and thepeptide treated with 20 mL liquid HF at −20° C. for 2 hours. After theevaporation of the HF under vacuum, the anisole was washed with ether(20 mL 5 times) and the solid residue dried in vacuo. The peptide wasextracted from the resin with TFA (10 mL, 3 times) and the TFAevaporated under vacuum. The residue was dissolved in 20 mL 30% AcOH andlyophilized. This process was repeated 3 times. The crude peptide waspurified by semiprep HPLC (Method H). The final product was obtained aswhite powder by lyophilization from dioxane, which gave 48 mg (64%) ofthe title compound.

HPLC (Method G) RT 27.70 minutes, 93%

TOF MS: 1424.6 (M⁺)

AAA in agreement with the title compound

TABLE 4 PROCEDURE FOR 0.05 mMOLE SCALE VOL- RE- STEP SOLVENT/ UME TIMEPEAT NO. REAGENT (ML) (MIN) (XS) COMMENT 1 NMP 5 120  1 Swells resin 2Ac₂O/PP/NMP 5 30  1 Protecting of side chain 3 NMP 5 2 6 check fornegative nin. 4 DIC/HOBT/NMP 5 30  1 Activation of COOH side chain 5MeNH₂/EtOH/ 5 60  1 Protecting of side chain 6 NMP 5 2 6 7 Pip/NMP 20% 510  1 Deprotection 8 Pip/NMP 20% 5 10  1 9 NMP 5 2 6 Check for positivenin. 10  AdacOH/BOP/ 5 2 1 NMP 11  NMP 5 2 6 Check for negative nin. 12 DCM 5 2 4

EXAMPLE 3H-D-Arg-Arg-cyclo(N^(α)(1-(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OHSTAGE 1

Fmoc-Phe-Arg(Tos)-O-resin→Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

Fmoc-Phe-Arg(Tos)-O-resin prepared from Boc-Arg(Tos)-O-Resin (0.3 g, 0.1mmole) (Example 1, Stage 1) was subjected to two deprotection of theFmoc protecting group by 20% piperidine in NMP (Table 1, steps 11-13).After washing and ninhydrin test (Method J), coupling of Fmoc-D-Phe wasachieved as described in Stage 1 (Example 1) (Table 1 steps 9-10) usingFmoc-D-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) andDIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1, steps 11-13). After washing andninhydrin test (Method J), coupling of Fmoc-Ser(BzL) was achieved asdescribed in Stage 1 (Example 1) (Table 1 steps 9-10) usingFmoc-Ser(Bzl) (0.25 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) andDIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc groupdeprotected as described above (Table 1, steps 11-13). After washing andpicric acid test (Method K), coupling of Fmoc-N^(α)(3-Boc aminopropylene)glycine was achieved as described in Table 1, steps 9-10 usingFmoc-N^(α)(3-Boc amino propylene)Gly (0.272 g, 0.6 mmole), BOP reagent(0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin waswashed and subjected to three deprotection of the Fmoc protecting groupby 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test(Method K) was performed. If the test did not show 98±2% deprotectionthe peptide resin was subjected again to 3 deprotection steps (Table 2,steps 1-2), washing and picric acid test (Method K). Coupling ofFmoc-L-Hyp(OBzl) was achieved in NMP by the addition of Fmoc-L-Hyp(OBzl)(0.33 g, 0.6 mmole) and after 5 minutes of shaking, solid PyBrOP reagent(0.28 g, 0.6 mmole) was added to the flask. After shaking for 10minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambienttemperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Method K) (Table 2, steps 3-6). If thetest did not show 98±2% coupling the peptide resin was subjected againto a third coupling for 2 hours at 50° C. (Table 2, step 7). The resinwas washed subjected to three deprotection of the Fmoc protecting groupby 20% Pip in NMP (Table 2, steps 1-2). After washing picric acid test(Method K) was performed. If the picric acid test did not show 98±2%deprotection, the resin was subjected again to deprotections steps(Table 2, steps 1-2). Coupling of Fmoc-Phe was achieved in NMP by theaddition of Fmoc-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and afterpicric acid test (Method K) the Fmoc group deprotected as describedabove (Table 2, steps 1-2). After washing picric acid test (Method K)was performed. If the test did not show 98±2% deprotection the peptideresin was subjected again to 3 deprotection steps (Table 2, steps 1-2),washing and picric acid test (Method K). Coupling of N^(α)(3-t-Bucarboxy propylene)Gly was achieved as described in Table 1 steps 9-10using N^(α)(3-t-Bu carboxy propylene)Gly (0.264 g, 0.6 mmole), BOPreagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resinwas then washed and subjected to the picric acid test (Method K). Afternegative test the resin was used for the next coupling.

STAGE 2

Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin→Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin

Fmoc-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Boc aminopropylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) wassubjected to three deprotection of the Fmoc protecting group by 20%Piperidine in NMP (Table 2, steps 1-2). After washing picric acid(Method K) test was performed. If the test did not show 98±2%deprotection the peptide resin was subjected again to 3 deprotectionsteps (Table 6, steps 1-2), washing and picric acid test. Coupling ofFmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6mmole) and after 5 minutes of shaking, solid PyBroP reagent (0.28 g, 0.6mmole) was added to the flask. After shaking for 10 minutes, the mixturewas adjusted to pH 8 by the addition of DIEA (0.209 mL, 1.2 mmole) andthe flask shaken for 2.5 hours at ambient temperature. The resin wasthen washed and subjected to a second coupling by the same procedure for20 hours. After washing the resin was subjected to picric acid test(Method K) (Table 2, steps 3-6). If the test did not show 98±2% couplingthe peptide resin was subjected again to a third coupling for 2 hours at50° C. (Table 2, step 7). The resin was washed subjected to threedeprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2,steps 1-2). After washing picric acid test (Method K) was performed. Ifthe test did not show 98±2% deprotection the peptide resin was subjectedagain to 3 deprotection steps (Table 2, steps 1-2), washing and picricacid test (Method K). Coupling of Fmoc-D-Arg(Tos) was achieved in NMP asdescribed in Stage 1 (Table 1, steps 9-10) using Fmoc-D-Arg(Tos) (0.33g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2mmole). The resin was washed 6 times with NMP (Table 1, step 15) andused in the next stages.

STAGE 3

Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin→H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl))Gly-Hyp-Phe-N^(α)(3-amido-propyl)Gly)-Ser-D-Phe-Phe-Arg-OH

Fmoc-D-Arg(Tos)-Arg(Tos)-N^(α)(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N^(α)(3-Bocamino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 2) wassubjected to deprotection of the Boc and t-Bu protecting groups and onresin cyclization according to Table 5. The peptide resin was washedwith DCM and deprotected as described in Stage 1 by 55% TFA in DCM.After washing and neutralization by 10% DIEA in NMP and washing 6 timeswith and NMP (Table 5, steps 1-5) the peptide was cyclized as follow:solid TBTU reagent (0.19 g, 6 mmole) was added to the flask. Aftershaking for 10 minutes, the mixture was adjusted to pH 8 by the additionof DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours atambient temperature. The resin was then washed and subjected to a secondcoupling by the same procedure for 20 hours. After washing the resin wassubjected to picric acid test (Method K) (Table 3, steps 8-11). If thetest did not show 98±2% cyclization the peptide resin was subjectedagain to a third cyclization for 2 hours at 50° C. (Table 2, step 12).The resin was washed, subjected to three deprotection of the Fmocprotecting group by 20% Pip in NMP (Table 5, steps 14-15). After washing6 times with NMP and 4 times with DCM, the resin was dried in vacuo for24 hours. The dried resin was subjected to HF as follows: to the drypeptide resin (0.4 g) in the HF reaction flask, anisole (2 mL) was addedand the peptide treated with 20 mL liquid HF at −20° C. for 2 hours.After the evaporation of the HF under vacuum, the anisole was washedwith ether (20 mL, 5 times) and the solid residue dried in vacuo. Thepeptide was extracted from the resin with TFA (10 mL 3 times) and theTFA evaporated under vacuum. The residue was dissolved in 20 mL 30% AcOHand lyophilized. This process was repeated 3 times. The crude peptidewas purified by semipreparative HPLC (Method H). The final product wasobtained as white powder by lyophilization from dioxane, which gave 59mg (34%) of the title compound.

[Table 5 follows at this point]

TABLE 5 PROCEDURE FOR 0.1 mMOLE SCALE STEP SOLVENT/ VOLUME TIME REPEATNO. REAGENT (ML) (MIN) (XS) COMMENT  1 DCM 10 2 3  2 TFA/DCM 10 2 1Deprotection 55%  3 TFA/DCM 10 20  1 Deprotection 55%  4 DCM 10 2 3  5NMP 10 2 4  6 DIEA/NMP 10 5 2 Neutralization 10%  7 NMP 10 2 5  8TBTU/NMP/ 10 150  3 Cyclization DIEA  9 NMP 10 2 4 Picric acid test. Ifless than 98 ± 2% Coupling perform Steps 10. 10 TBTU/NMP/ 10 20 h 3Cyclization, DIEA Check pH, adjust to pH 8 with DIEA. 11 NMP 10 2 4Picric acid test. If less than 98 ± 2% coupling perform Step 12. 12TBTU/NMP/ 10 120  3 Cyclization, 50° DIEA C. Check pH, adjust to pH 8with DIEA. 13 NMP 10 2 6 14 Piper- 10 10  1 Deprotection idine/ NMP 20%15 Piper- 10 1 idine/ NMP 20% 16 NMP 10 2 6 Check for positive nin. 17DCM 10 2 4

HPLC (Method G)RT 33.62 minutes (91%)

TOF MS: 1278 (M⁺)

AAA in agreement with the title compound

EXAMPLE 4H-D-Arg-Arg-cyclo(N^(α)(4-propanoyl)Gly-Hyp-Phe-N^(α)(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH

Title compound was synthesized according to Example 3 except that instage 1, Fmoc-N^(α)(3-Boc-amino-propylene)-S-Phe (0.326) was substitutedfor Fmoc-N^(α)(3-Boc amino propylene)Gly. A total of 0.643 gBoc-L-Arg(Tos)-O-resin (0.39 meq/g, 0.250 mmole) was used and reagentquantities were adjusted accordingly. Cyclic peptide yield (from halfthe total resin used) was 74 mg (42%) of the title compound.

SPECIFIC EXAMPLES OF BUILDING UNITS

The following specific examples of novel building units are provided forillustrative purposes not meant to be limiting. The following isdescribed in sections, including “Procedures”, “Methods”, “Compounds”and “EXAMPLES”. “Procedures” are detailed stepwise descriptions ofsynthetic procedures according to the more general schemes. “Methods”are general descriptions of analyses used to determine the progress ofthe synthetic process. Numbered “Compounds” are either the startingmaterial or intermediates for further numbered “Compounds” the synthesisof which progresses according to the specified “Procedure.” Several“Compounds” used in series produce “EXAMPLES” of novel building units ofthe present invention. For instance, “Compounds” 27-29, 41-44, 46-47,51-55, 62, 63, 67 and 76-79 are actually “EXAMPLES” 5-25, respectively,of novel building units of the present invention. “Compounds” 1-26,30-40, 45, 48-50, 56-61, 64-66, 68-75 are starting materials orintermediates (only) for the synthesis of “EXAMPLES”.

Procedure 1

Synthesis of N-Boc alkylene diamines (BocNH(CH₂)_(n)NH₂) (knowncompounds).

To a solution of 0.5 mole alkylene diamine in 0.5 L CHCl₃ cooled in anice-water bath, was added dropwise, with stirring, a solution of 10.91 g(0.05 mole) Boc₂O in 0.25 L CHCl₃ for 3 h. The reaction mixture wasstirred for 16 h. at room temperature and then washed with water (8×250mL). The organic phase was dried over Na₂SO₄ and evaporated to drynessin vacuo.

Procedure 2

Synthesis of N-Boc, N-Bzl alkylene diamines (BocNH(CH₂)_(n)NH-Bzl).

To a solution of 0.05 mole of mono Boc alkylene diamine in 60 mL MeOHwas added 2.77 mL (0.02 mole) Et₃N, 9.02 g (0.075 mole) MgSO₄, and 5.56mL (0.055 mole) of freshly distilled benzaldehyde. The reaction mixturewas stirred under room temperature for 1.5 h. Then 11.34 g (0.3 mole) ofNaBH₄ were added in small portions during 0.5 h with cooling to −5° C.The reaction mixture was then stirred for 1 h at −5° C. and for another1 h at 0° C. The reaction was stopped by addition of 200 mL water andthe product was extracted with EtOAc (3×200 mL). The combined EtOAcextracts were washed with water (4×100 mL). The organic phase wasextract with 0.5 N HCl (4×100 mL) and the aqueous solution wasneutralized under cooling with 25 mL 25% NH₄OH, extracted with CHCl₃(3×100 ml) and the combined extracts were washed with water (3×80 mL),dried over Na₂SO₄ and evaporated to dryness in vacuo.

Procedure 3

Synthesis of (R) or (S) α-hydroxy acids (known compounds).

To a solution of 16.52 g (0.1 mole) (R) or (S) amino acid in 150 ml 1NH₂SO₄ was added dropwise a solution of 10.35 g (0.15 mole) NaNO₂ in 100mL H₂O during 0.5 h with stirring and cooling in an ice bath. Thereaction mixture was stirred 3 h. at 0° C. and additional 18 h at roomtemperature, then the (R)- or (S)- hydroxy acid was extracted with etherin a continuous ether extractor. The etheral solution was washed with 1NHCl (2×50 mL), H₂O (3×80 mL), dried over Na₂SO₄ and evaporated todryness. The product was triturated twice from ether:petrol-ether(40-60° C.) (1:10). The precipitate was filtered, washed with 50 mLpetrol-ether and dried.

Procedure 4

Synthesis of (R) or (S) α-hydroxy acid methyl esters (known compounds).

To a suspension of 0.065 mole (R)- or (S)- hydroxy acid in 100 mL etherwas added under cooling in an ice bath 300 mL of an etheral solution ofCH₂N₂ until stable yellow color of reaction mixture was obtained. Thenthe ether solution was washed with 5% KHCO₃ (3×100 mL) and H₂O (2×80mL), dried over Na₂SO₄ and evaporated to dryness. The product was driedin vacuo.

Procedure 5

Synthesis of triflate of (R) or (S) α-hydroxy acid methyl esters.

To a cooled solution of 2.67 ml (0.033 mole) pyridine in 20 mL dry DCMwas added 5.55 mL (0.033 mole) Trf₂O at −20° C. (dry ice in EtOH bath),then after 5 min a solution of 0.03 mole (R) or (S) α-hydroxy acidmethyl ester in 20 mL dry DCM was added dropwise. The reaction mixturewas stirred at room temperature for 45 min, then was passed through ashort silica gel column (2 cm). The product was eluted with 400 mL ofpetrol-ether:methylene-chloride (1:1). The solvent was evaporated invacuo.

Procedure 6

Synthesis of (R) or (S) N^(α)(Bzl)(N^(ω)-Boc-amino alkylene)amino acidmethyl esters ((R) or (S) BocNH(CH₂)_(n)N(Bzl)CH(R)COOMe).

To a solution of 0.022 mole of N^(α)-Boc, N^(ω)-Bzl alkylene diamine in20 mL of dry DCM was added 3.04 mL (0.022 mole) Et₃N. Then a solution of0.02 mole of (R) or (S) α-hydroxy acid methyl ester triflate in 25 mLdry DCM was added dropwise (0.5 h.) under cooling in an ice-water bath.The reaction mixture was stirred at room temperature for 18 h. Then 150mL of CHCl₃ was added and the yellow solution was washed with water(3×80 mL). The organic phase was dried over Na₂SO₄ and adsorbed onsilica-gel and dried in vacuo. The silica-gel was washed on filter with0.5 L of petrol-ether and with 0.5 L of 2% EA in PE. Then the productwas eluted from silica with 0.5 L of mixture petrol-ether:ethyl-acetate(4:1). The solvent was evaporated in vacuo. If the product was not cleanit was further purified on a small column of silica-gel (250 mL). Thefirst impurities were eluted with 0.8 L of hexane then the product waseluted with 1.5 L of mixture of petrol-ether:ethyl-acetate (4:1).

Procedure 7

Hydrolysis of methyl esters

To a solution of 0.015 mole of methyl ester in 40 mL MeOH was added 10mL 7.5 N NaOH cooled in an ice-water bath. The reaction mixture wasstirred at room temperature for approximately 24 h (until the methylester spot disappears on TLC). Then 100 mL of water were added and thereaction mixture was washed with petrol-ether (3×80 mL). The aqueoussolution was acidified under cooling by addition of 40 mL 2N HCL. Theproduct was extracted with a mixture of CHCl₃:i-PrOH (3:1) (3×80 mL),dried over Na₂SO₄, evaporated to dryness and dried in vacuo to obtain awhite foam in quantitative yield.

Procedure 8

Removal of Bzl by Hydrogenation with Pd/C

To a solution of 0.012 mole (R) or (S) N^(α)(Bzl)(N^(ω)-Boc-aminoalkylene) amino acid in 60 mL MeOH—DMF (11-1) was added 0.5 g 10% Pd/C.The solution was hydrogenated for 4 h under a pressure of 45-50 Psi atroom temperature. Then 200 mL of a mixture of DMF:MeOH:H₂O:glacial AcOH(1:3:5:1) was added. The catalyst was filtrated off and washed (is theacetic acid?) with H₂O or MeOH (2×15 mL). The combined filtrate wasevaporated to dryness and recrystallized from methanol:ether (15mL:250). The precipitate was filtered and dried in vacuo.

Procedure 9

Synthesis of (R) or (S) N^(α)(Fmoc)(N^(ω)-Boc-amino alkylene) aminoacid.

To 50 mL water was added 0.07 mole of (R) or (S) N^(α) (N^(ω)-Boc-aminoalkylene) amino acid and 1.95 mL (0.014 mole) Et₃N. The suspension wasstirred 2-3 h until a clear solution was obtained. Then a solution of2.25 g (0.07 mole) of FmocOSu in 100 mL ACN was added. The reactionmixture was stirred 18 h at room temperature, then 150 mL water wasadded and the solution was washed with petrol-ether (3×100 mL) and withether:petrol-ether (1:4). The aqueous solution was acidified by additionof 14 mL 1N HCL. The product was extracted with EtOAc (4×100 mL) and theorganic phase was washed with 0.5 N HCl (2×50 mL), H₂O (3×80 mL), driedover Na₂SO₄, evaporated to dryness and recrystallized fromether:petrol-ether (80 mL:200 mL)

Procedure 10

Synthesis of S-benzylcysteamine (Bzl-S—(CH₂)₂—NH₂) (known compound).

To a suspension of 0.1 mole cysteamine hydrochloride in 20 mL methanolwere added 13.6 mL of 25% ammonia solution, followed by dropwiseaddition of 0.12 mole benzyl bromide at room temperature. The mixturewas stirred for 0.5 h, and the formed precipitate ofS-dibenzylcysteamine was collected by filtration. The product wasextracted with ether (3×100 mL) and the organic phase was successivelywashed with brine (2×100 mL), dried over MgSO₄ and the solventevaporated in vacuo. The crude product was essentially pure enough forthe next step. It could, however be recrystallized from ethyl acetate.Yield 86%, of white solid. m.p. 85-6° C. NMR (CDCl₃) in agreement withthe title compound.

Procedure 11

Synthesis of N^(α)-(ω-(benzylthio)alkylene)phthalimides((Bzl-S—(CH₂)_(n)—N═Pht)) (known compounds)

N-(ω-bromoalkylene)phthalimide (0.1 mole) and benzyl mercaptane (0.11mole) were stirred with 0.1 mole potassium carbonate in 100 mL DMSO at50° C. for 24 hours. The mixture was poured into ice-water and theproduct was allowed to crystallize for 0.5 hour, collected by filtrationand recrystallized from i-PrOH.

Procedure 12

Synthesis of N^(α)-(ω-(benzylthio)alkyl)amines (Bzl-S—(CH₂)_(n)—NH₂)(known compounds).

Hydrazinolysis of the phthalimide group was performed by refluxing 0.09mole of N^(α)-(ω-(benzylthio)alkylene)-phthalimide (Procedure 11) with120 mL 1M solution of hydrazine hydrate in ethanol (diluted withadditional 220 mL ethanol) for 2 hours. The formed precipitate wascollected by filtration and hydrolyzed with 180 mL 2N HCl at 50° C. for0.5 hour. The water was evaporated in vacuo and the crude hydrochloridedissolved in 50 mL 25% ammonia solution. The free amine was extractedwith DCM (4×100 mL) and the organic phase was washed with brine (2×100mL), dried over MgSO₄ and the solvent evaporated in vacuo. The crudeN-(ω-(benzyl-thio)alkyl)amine was distilled under reduced pressure, andappeared as colorless oil, which could be kept refrigerated undernitrogen for prolonged periods.

Procedure 13

Synthesis of (R) and (S) N^(α)-(ω-(benzylthio)alkylene)amino acidsmethyl esters ((R) or (S) (Bzl-S—(CH₂)_(n)—NH—CH(R)—COOMe)).

To a solution of 15 mmol N-(ω-(benzylthio)alkyl)amine and 15 mmol DIEAin 55 mL DCM was added dropwise a solution of 15 mmol (R) or (S)α-hydroxy acid methyl ester triflate (Procedure 5) in 55 mL DCM at 0° C.The reaction mixture was then stirred at room temperature for 18 h. Themixture was then diluted with 100 mL DCM and washed with water (3×100mL). The crude product was cleaned on a silica-gel column withDCM:MeOH(99:1), and was further crystallized from DIE:hexane.

In the case of Glycine, bromoacetic acid esters were suitable startingmaterials. Identical results were obtained when both these substrateswere reacted with N-(ω-(benzylthio) alkyl)amines.

Procedure 14

Synthesis of (R) or (S) Boc-N^(α)-(ω-(benzylthio)alkylene)amino acids((R) or (S) (Bzl-S—(CH₂)_(n)—N(Boc)—CH(R)—COOH)).

10 mmol (R) or (S) N-(ω-(benzylthio)alkylene) amino acid methyl esterwas dissolved in 50 mL 1,4-dioxane and 50 mL 1N NaOH were added. Themixture was stirred at room temperature overnight. The disappearance ofthe starting material was followed by TLC (Silica gel+F₂₅₄,CHCl₃:MeOH-1:4). When all the ester was hydrolyzed, 50 mL water wereadded, followed by 30 mmol Boc₂O. The mixture was stirred overnight,then the dioxane was evaporated in vacuo, the mixture was cooled in anice-water bath, covered with 100 mL EtAc and acidified with saturatedKHSO₄ to pH 2-3. The layers were separated, and the aqueous layer wasextracted with additional 2×100 mL EtOAc. The organic layer was washedwith water (2×100 mL), dried over MgSO₄ and the solvent evaporated invacuo. The crude product was cleaned on a silica-gel column withDCM:MeOH-99:1 or crystallized from DIE:hexane.

Procedure 15

Synthesis of dipeptides (S,S)-Boc-aminoacid-N^(α)-(ω-(benzylthio)alkylene)amino acids esters.

A solution of 1.1 mmol Boc-amino acid, 1 mmolN^(α)-(ω-(benzylthio)alkylene) amino acid ester, 1.1 mmol BOP and 3 mmolDIEA in 10 mL DCM was stirred at room temperature for 2 hours. Then themixture was diluted with 40 mL of DCM and washed successively withsaturated KHSO₄ (3×100 mL), saturated KHCO₃ (3×100 mL) and brine (2×100mL), dried over MgSO₄ and the solvent evaporated in vacuo. The crudeproduct was cleaned on a silica-gel column with DCM:MeOH-99:1 orcrystallized from DIE:hexane.

Procedure 16

Synthesis of N^(α)-Bzl ω-amino acids t-butyl esters(Bzl—NH—(CH₂)_(n)—COO-t-Bu).

A solution of 0.05 mole of amino acid t-butyl ester acetate in 200 mLH₂O was acidified to pH 2 with AcOH, washed with PE (70 mL X5) cooledand the pH adjusted to 9 by NH₄OH 25%. The free amino acid t-butyl esterwas extracted with i-Pr:CHCl (3:1, 3×100 mL). The combined extracts weredried on Na₂SO₄ and evaporated to dryness under vacuum. The benzylationreaction was performed according to Procedure 2.

Procedure 17

Synthesis of N^(α)-(Bzl)(ωt-Bu carboxy alkylene)glycine Bzl ester(N-(Bzl) (CH₂)_(n)—COO-t-Bu)CH₂—COO-Bzl)

To a stirred solution of 0.015 mole of N-Bzl ω-amino acid t-butyl esterin 10 mL DMF at 0° C. were added 2.61 mL of DIEA and 2.38 mL of benzylbromo acetate. The reaction mixture was stirred 30 min at 0° C. and 3 hat room temperature. After the addition of 200 mL of ether, theprecipitate was removed by filtration and the organic phase washed withH₂O (3×80 mL), 1N HCl (3×80 mL), H₂O (3×80 mL), dryed over Na₂SO₄ andevaporated to dryness under vacuum. The resulting oil was dried undervacuum.

METHODS

ANALYTICAL TLC was performed on TLC plates of silica gel E (Merck F₂₅₄),using the following solvent systems:

METHOD A DCM:MeOH:AcOH 16:4:0.5 METHOD B PE:EtOAc 1:1 METHOD C PE:EtOAc4:1 METHOD D CHCl₃:EtOAc 4:1 METHOD E PE:EtOAc 9:1 METHOD F CHCl₃:EtOAc19:1

METHOD G ANALYTICAL REVERSE PHASE HPLC

Column Merck LICHROCART RP-18 5 μm, 250 × 4 mm. Mobile Phases A = 0.1%TFA in H₂O B = 0.1% TFA in ACN Gradient T = 0-5 min A(75%), B(25%) T =30 min A(50%), B(50%) T = 40 min A(100%), B(0%) T = 50 min A(100%),B(0%) Flow = 1 mL/minute Temperature = 23° C.

METHOD H—SEMIPREPARATIVE REVERSE PHASE HPLC

The crude peptide was dissolved in MeOH (1 mL) and chromatographed usingreverse phase semipreparative HPLC with the following conditions:

Column Merck Hibar LICHROSORB RP-18 7 μm, 250 × 410 mm Mobile Phases A =0.1% TFA in H₂O B = 0.1% TFA in ACN Gradient T = 0-10 min A(80%), B(20%)T = 60 min A(100%), B(0%) T = 70 min A(100%), B(0%) Flow = 4mL/minute Temperature = 23° C.

METHOD J NINHYDRIN TEST (NIN. TEST)

The test was performed according to Kaiser et al. Anal. Biochem., 34:595(1970) which is incorporated herein by reference in its entirety. Testwas considered negative when the resin did not change color afterheating to 11° C. for 2 minutes with the test mixture. Test wasconsidered positive or slightly positive when the resin was dark orfaint purple after heating to 110° C. for 2 minutes with the testmixture.

METHOD K—QUANTITATIVE PICRIC ACID TEST

Picric acid test was performed after removal of the Fmoc protectinggroup from the amino acid preceding the coupling of protectedNα(ω-alkylene) building unit. This absorbance was taken as 100% freeamines. After coupling the test was used to check yield of % coupling bycomparison.

The resin 0.1 mmole was treated according to steps 1-8, Table 1. Afterstep 8 the resin was introduced into a centrifuge tube and shaken with40 mL of 5% DIEA:95% DCM for ten minutes. The resin was centrifuged 5minutes and 4 mL of the solution were pipette into 40 mL. EtOH and theabsorbance at 358 nm measured. This procedure was repeated 3 times andthe average absorbance calculated.

TABLE 6 PROCEDURE FOR 0.1 mMOLE SCALE STEP SOLVENT/ VOLUME TIME REPEATNO. REAGENT (ML) (MIN) (XS) COMMENT 1 NMP 10 1 3 2 DCM 10 1 2 3 Picricacid 10 1 2 0.05M/DCM 4 DMF 10 0.5 10  5 NMP 10 1 2 6 NMP 10 2 3 7 10%EtOH/DCM 10 1 1 8 DCM 10 1 2

COMPOUND 1

N-Boc diamino ethane (known compound)

A solution of 33.4 mL of ethylene diamine in CHCl₃ and 10.91 g of Boc₂Owere used (Procedure 1). Yield 97% of colorless oil.

TLC (Method A) Rf 0.2-0.24 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 2

N-Boc 1,3 diamino propane (known compound).

A solution of 41.7 mL of 1,3 diamino propane in CHCl₃ and 10.91 g ofBoc₂O were used (Procedure 1). Yield 96% of colorless oil.

TLC (Method A) Rf 0.27-0.3 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 3

N-Boc 1,4 diamino butane (known compound).

A solution of 44.08 g of 1,4 diamino butane and 10.91 g of Boc₂O wereused (Procedure 1). Yield 98% of white oil.

TLC (Method A) Rf 0.32-0.35 (one spot); NMR (CDCl₃) in agreement withthe title compound.

COMPOUND 4

N-Boc 1,6 diamino hexane (known compound).

A solution of 58.10 g of 1,6 diamino hexane in CHCl₃ and 10.91 g ofBoc₂O were used (Procedure 1). Yield 70% of colorless oil afterpurification on a silica gel column and elution with CHCl₃—MeOH (4:1).

TLC (Method A) Rf 0.50-0.54 (one spot); NMR (CDCl₃) in agreement withthe title compound.

COMPOUND 5

N-Boc, N-Bzl 1,2 diamino ethane

A solution of 8.01 g of Boc ethylene diamine (COMPOUND 1) was used(Procedure 2). Yield 65% of colorless oil.

TLC (Method A) Rf 0.62-0.65 (one spot); NMR (CDCl₃) in agreement withthe title compound.

COMPOUND 6

N-Boc, N-Bzl, 1,3 diamino propane.

A solution of 8.71 g of N-Boc 1,3 diamino propane (COMPOUND 2) was used(Procedure 1). Yield 75% of colorless.

TLC (Method A) Rf 0.63-0.68 (one spot); NMR (CDCl₃) in agreement withthe title compound.

COMPOUND 7

N-Boc, N-Bzl 1,4 diamino butane

A solution of 9.41 g of N-Boc 1,4 diamino butane (Compound 3) was used(Procedure 1). Yield 63% of white oil.

TLC (Method A) Rf 0.65-0.72 (one spot); NMR (CDCl₃) in agreement withthe title compound.

COMPOUND 8

N-Boc, N-Bzl 1,6 diamino hexane

A solution of 10.82 g of N-Boc 1,6 diamino hexane (Compound 4) was used(Procedure 1). The ethyl acetate solution after extraction was driedover Na₂SO₄ and evaporated under vacuum to dryness. The remaining crudeproduct was dissolved in 400 mL chloroform and washed with 0.5 N HCl(3×80 mL, 0.12 mole), water (2×100 mL) dried over Na₂SO₄ and evaporatedto dryness. Then 200 mL of ether was added. The precipitate wasfiltered, washed with ether (3×50 mL) and dried under vacuum.

Yield 70% of white solid mp 150-152° C.; TLC (Method A) Rf 0.8 (onespot).

To remove HCl from product with n=6, the HCl salt was dissolved inCHCl₃, washed with an alkali solution (0.5% NH₄OH), dried over Na_(≦)SO₄and evaporated to dryness. NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND 9

(S)-3-Phenylacetic acid methyl ester (known compound)

A suspension of 10.8 g of (S)-3-Phenylacetic acid in 100 mL ether wastreated with diazomethane (Procedure 4).

Yield 85%. TLC (Method B) Rf 0.6-0.65 (one spot); (α)_(D)=+3,3 (c=1,MeOH); NMR (CDCl₃) in agreement with the title compound.

COMPOUND 10

(R)-3-Phenylacetic acid methyl ester (known compound).

A suspension of 10.8 g (R)-3-Phenylacetic acid in 100 mL ether wastreated with diazomethane (Procedure 4). Yield 84%.

TLC (Method B) Rf 0.6-0.65 (one spot); (α)_(D)=−3,3 (c=1, MeOH); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 11

(S)-O-Trf-3-Phenylacetic acid methyl ester

To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), asolution of 5.4 g of (S)-3-Phenylacetic acid methyl ester was added.After the workup (Procedure 5), the yield was 74%. The product was usedimmediately or kept in a cold desiccator under Ar.

COMPOUND 12

(R)-O-Trf-3-Phenylacetic acid methyl ester

To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), asolution of 5.4 g of (R)-3-Phenylacetic acid methyl ester was added.After the workup (Procedure 5), the yield was 74%. The product was usedimmediately or kept in a cold desiccator under Ar.

COMPOUND 13

N^(α)(Bzl)(2-Boc-amino ethylene)(R)Phenylalanine methyl ester

A solution of 6.24 g of (S)-O-Trf-3-Phenyllactic acid methyl ester indry DCM (Compound 11) was added to a solution of 5.51 g of N-Boc,N-Bzl-diamino ethane (Compound 5) in dry DCM (Procedure 6). Yield 69.2%

(α)_(D)=+64.0 (c=1, MeOH); TLC (Method C) Rf=0.41 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 14

N^(α)(Bzl)(3-Boc-amino propylene)(S)Phenylalanine methyl ester

A solution of 6.24 g of (R)-O-Trf-3-Phenyllactic acid methyl ester indry DCM (Compound 12) was added to a solution of 5.82 g of N-Boc,N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 67.7%

(α)_(D)=−55.8 (c=1, MeOH); TLC (Method C) Rf=0.38 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 15

N^(α)(Bzl)(4-Boc-amino butylene)(S)Phenylalanine methyl ester

A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester indry DCM (Compound 12) was add to a solution of 6.12 g of N-Boc,N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 58.6%

(α)_(D)=−62.6 (c=1, MeOH); Rf (Method C) 0.42 (one spot); NMR (CDCl₃) inagreement with the title compound.

COMPOUND 16

N^(α)(Bzl)(6-Boc-amino hexylene)(S)Phenylalanine methyl ester

A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester indry DCM (Compound 12) was add to a solution of 6.74 g of N-Boc,N-Bzl-diamino hexane (Compound 8) in dry DCM (Procedure 6). Yield 78.9%

(α)_(D)=−60.0 (c=1, MeOH); TLC (Method C) Rf=0.47 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 17

N^(α)(Bzl)(3-Boc-amino propylene)(R)Phenylalanine methyl ester

A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester indry DCM (Compound 11) was add to a solution of 5.82 g of N-Boc,N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 51.5%

(α)_(D)=+58.8 (c=1, MeOH); TLC (Method C) Rf=0.35 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 18

N^(α)(Bzl)(4-Boc-amino butylene)(R)Phenylalanine methyl ester

A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester indry DCM (Compound 11) was add to a solution of 6.12 g of N-Boc,N-Bzl-diamino butane (Compound 7) in dry DCM (Procedure 6). Yield 66.8%

(α)_(D)=+59.0 (c=1, MeOH); TLC (Method C) Rf=0.33 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 19

N^(α)(Bzl)(3-Boc-amino propylene)(S)Phenylalanine

A solution of 6.61 g of N^(α)(Bzl)(3-Boc-amino propylene)(S)Phenylalanine methyl ester in MeOH (Compound 14) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 89.5%

(α)_(D)=−24.0 (c=1, MeOH); TLC (Method B) Rf=0.16 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 20

N^(α)(Bzl)(4-Boc-amino butylene)(S)Phenylalanine

A solution of 6.61 g of N^(α)(Bzl)(4-Boc-amino butylene)(S)Phenylalanine methyl ester in MeOH (Compound 15) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 73.5%

(α)_(D)=−12.0 (c=1, MeOH); TLC (Method D) Rf=0.6 (one spot); NMR (CDCl₃)in agreement with the title compound.

COMPOUND 21

N^(α)(Bzl)(3-Boc-amino propylene)(R)Phenylalanine

A solution of 6.40 g of N^(α)(Bzl)(3-Boc-amino propylene)(R)Phenylalanine methyl ester in MeOH (Compound 17) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 100%

(α)_(D)=+15.33 (c=1, MeOH); TLC (Method B) Rf=0.38 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 22

N^(α)(Bzl)(4-Boc-amino butylene)(R)Phenylalanine

A solution of 6.61 g of N^(α)(Bzl)(4-Boc-amino butylene)(R)Phenylalanine methyl ester in MeOH (Compound 18) was hydrolyzed byNaOH 7.5N (Procedure 7). Yield 100%

(α)_(D)=+12.0 (c=1, MeOH); TLC (Method D) Rf=0.54 (one spot); NMR(CDCl₃) in agreement with the title compound.

COMPOUND 23

N^(α) (3-Boc-amino propylene)(S)Phenylalanine HCl

A solution of 5.39 g of N^(α)(Bzl)(3-Boc-amino propylene)(S)Phenylalanine (Compound 19) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 86.1%

TLC (Method A) Rf=0.51 (one spot); NMR (D₂O+Na₂CO₃) in agreement withthe title compound.

COMPOUND 24

N^(α) (4-Boc-amino butylene)(S)Phenylalanine HCl

A solution of 5.56 g of N^(α)(Bzl)(4-Boc-amino butylene)(S)Phenylalanine (Compound 20) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 79.25%

TLC (Method A) Rf=0.50 (one spot); NMR (D₂O+Na₂CO₃) in agreement withthe title compound.

COMPOUND 25

N^(α) (3-Boc-amino propylene)(R)Phenylalanine HCl

A solution of 5.39 g of N^(α)(Bzl)(3-Boc-amino propylene)(R)Phenylalanine (Compound 21) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 75.5%

TLC (Method A) Rf=0.51 (one spot); NMR (D₂O+Na₂CO₃) in agreement withthe title compound.

COMPOUND 26

N^(α) (4-Boc-amino butylene)(R)Phenylalanine HCl

A solution of 5.56 g of N^(α)(Bzl)(4-Boc-amino butylene)(R)Phenylalanine (Compound 22) in MeOH-DMF was hydrogenated on Pd/C(Procedure 8). Yield 73.85%

TLC (Method A) Rf=0.50 (one spot); NMR (D₂O+Na₂CO₃) in agreement withthe title compound.

EXAMPLE 5 COMPOUND 27

N^(α)(Fmoc)(3-Boc-amino propylene)(S)Phenylalanine

A solution of 2.51 g of N^(α) (3-Boc-amino propylene)(S)Phenylalanine.HCl (Compound 23) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 64.84%

TLC (Method D) Rf=0.74 (one spot); (α)_(D)=−87 (c=1, MeOH); HPLC (MethodG) 92%; NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 6 COMPOUND 28

N^(α)(Fmoc)(3-Boc-amino propylene)(R)Phenylalanine

A solution of 2.51 g of N^(α) (3-Boc-amino propylene)(R)Phenylalanine.HCl (Compound 25) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 61.56%

TLC (Method D) Rf=0.62 (one spot); (α)_(D)=+79.6 (c=1, MeOH); HPLC(Method G) 94%; NMR (CDCl₃) in agreement with the title compound.

EXAMPLE 7 COMPOUND 29

N^(α)(Fmoc)(4-Boc-amino butylene)(S)Phenylalanine

A solution of 2.61 g of N^(α) (4-Boc-amino butylene)(S)Phenylalanine.HCl (Compound 24) in H₂O-ACN was reacted with FmocOSu(Procedure 9). Yield 56%

TLC (Method D) Rf=0.64 (one spot); HPLC (Method G) 89%; NMR (CDCl₃) inagreement with the title compound.

COMPOUND 30

O-Trf-(S)-Lactic acid methyl ester

To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5), asolution of 2.9 mL of (S)lactic acid methyl ester was added. After theworkup (Procedure 5), the yield was 70%. The product was usedimmediately or kept in a cold desiccator under Ar.

COMPOUND 31

O-Trf-(R)-Lactic acid methyl ester

To a cooled solution of Trf₂O and pyridine in DCM (Procedure 5), asolution of 2.9 mL of (R) lactic acid methyl ester was added. After theworkup (Procedure 5), the yield was 70%. The product was usedimmediately or kept in a cold desiccator under Ar.

COMPOUND 32

N^(α)(Bzl)(3-Boc-amino propylene)(S)Alanine methyl ester

A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM(Compound 31) was add to a solution of 5.82 g of N-Boc, N-Bzl-diaminopropane (Compound 6) in dry DCM (Procedure 6). Yield 69.5%

(α)_(D)=−6.6 (C=1, MeOH); TLC (Method C) Rf=0.42 (one spot); TLC (MethodD) Rf=0.92 (one spot); TLC (Method E) Rf=0.13 (one spot); NMR (CDCl₃) inagreement with the title compound.

COMPOUND 33

N^(α)(Bzl)(3-Boc-amino propylene)(R)Alanine methyl ester

A solution of 4.72 g of O-Trf-(S-lactic acid methyl ester in dry DCM(Compound 30) was add to a solution of 5.82 g of N-Boc, N-Bzl-diaminopropane (Compound 6) in dry DCM (Procedure 6). Yield 71%

(α)_(D)=+6.53 (C=1, MeOH); TLC (Method C) Rf=0.42 (one spot); TLC(Method D) Rf=0.93 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND 34

N^(α)(Bzl)(6-Boc-amino hexylene)(S)Alanine methyl ester

A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM(Compound 31) was add to a solution of 6.74 g of N-Boc, N-Bzl-diaminohexane (Compound 8) in dry DCM (Procedure 6). Yield 81.42%

(α)_(D)=−6.76 (C=1, MeOH); TLC (Method D) Rf=0.95 (one spot); TLC(Method E) Rf=0.26 (one spot); NMR (CDCl₃) in agreement with the titlecompound.

COMPOUND 35

N^(α)(Bzl)(2-Boc-amino propylene)(S)Alanine

A solution of 5.25 g of N^(α)(Bzl)(2-Boc-amino ethylene) (S)Alaninemethyl ester in MeOH (Compound 32) was hydrolyzed by NaOH 7.5N(Procedure 7). Yield 100% of white solid, mp 64° C.

(α)_(D)=+0.5 (C=1, MeOH); TLC (Method A) Rf=0.64 (one spot); TLC (MethodD) Rf=0.47 (one spot); NMR (CDCl₃) in agreement with the title compound.

COMPOUND 36

N^(α)(Bzl)(6-Boc-amino hexylene)(S)Alanine

A solution of 5.88 g of N^(α)(Bzl)(6-Boc-amino hexylene) (S)Alaninemethyl ester (Compound 34) in MeOH was hydrolyzed by NaOH 7.5N(Procedure 7). Yield 100%

(α)_(D)=+0.7 (C=1, MeOH); TLC (Method D) Rf=0.51 (one spot); NMR (CDCl₃)in agreement with the title compound.

COMPOUND 37

N^(α)(Bzl)(3-Boc-amino propylene)(R)Alanine

A solution of 5.25 g of N^(α)(Bzl)(2-Boc-amino ethylene) (R)Alaninemethyl ester (Compound 35) in MeOH was hydrolyzed by NaOH 7.5N(Procedure 7). Yield 100%

(α)_(D)=−0.5 (C=1, MeOH); TLC (Method D) Rf=0.51 (one spot); NMR (CDCl₃)in agreement with the title compound.

COMPOUND 38

N^(α) (3-Boc-amino propylene)(S)Alanine.HCl

A solution of 4.47 g of N^(α)(Bzl)(3-Boc-amino propylene) (S)Alanine(Compound 35) in MeOH was hydrogenated on Pd/C (Procedure 8). Yield 75%

TLC (Method A) Rf=0.42 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 39

N^(α) (6-Boc-amino hexylene)(S)Alanine. HCl

A solution of 5 g of N^(α)(Bzl)(4-Boc-amino hexylene) (S)Alanine(Compound 36) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield64.5% of white solid, mp 134-136° C.

TLC (Method A) Rf=0.39 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 40

N^(α) (3-Boc-amino propylene)(R)Alanine. HCl

A solution of 5 g of N^(α)(Bzl)(4-Boc-amino propylene) (R)Alanine(Compound 37) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield79.1%.

TLC (Method A) Rf=0.39 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

EXAMPLE 8 COMPOUND 41

N^(α)(Fmoc)(3-Boc-amino propylene)(S)Alanine

A solution of 2.82 g of N^(α)(Bzl)(4-Boc-amino propylene) (S)Alanine.HCl (Compound 38) in H₂O-ACN was reacted with FmocOSu (Procedure 9).Yield 75% of white solid, mp 70-72° C.

TLC (Method D) Rf=0.65 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 66.40 6.78 5.63 Calc: 66.65 6.885.93

EXAMPLE 9 COMPOUND 42

N^(α)(Fmoc)(6-Boc-amino hexylene)(S)Alanine

A solution of 3.25 g of N^(α)(Bzl)(4-Boc-amino hexylene) (S)Alanine .HCl(Compound 39) in H₂O-ACN was reacted with FmocOSu (Procedure 9). Yield72.8% of white solid, mp 70-72° C.

TLC (Method D) Rf=0.7 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 68.37 7.40 5.23 Calc: 68.21 7.505.49

HPLC (Method G) 90%

EXAMPLE 10 COMPOUND 43

N^(α)(Fmoc)(3-Boc-amino propylene)(R)Alanine

A solution of 2.82 g of N^(α)(Bzl)(4-Boc-amino propylene) (S)Alanine.HCl (Compound 40) in H₂O-ACN was reacted with FmocOSu (Procedure 9).Yield 75.9% of white solid, mp 70-72° C.

TLC (Method D) Rf=0.5 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 66.4 6.78 5.63 Calc: 66.65 6.885.93

EXAMPLE 11 COMPOUND 44

N-(2-(benzylthio)ethylene)glycine ethyl ester

The title compound was prepared according to procedure 13 from ethylbromo acetate.

Yield 75% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-61.16, H-7.70, N-3.96; found:C-61.45, H-8.03, N-3.49.

COMPOUND 45

N-(3-(benzylthio)propylene)glycine methyl ester

The title compound was prepared according to procedure 13 from methylbromo acetate.

Yield 74% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound.

EXAMPLE 12 COMPOUND 46

N-(2-(benzylthio)ethylene)(S)leucine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (R) leucine methyl ester (Procedure 5).

Yield 70% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-65.05, H-8.53, N-4.74; found:C-66.29, H-9.03, N-4.49. (a)_(D14)=−51.2° C. (C 0.94,DCM).

EXAMPLE 13 COMPOUND 47

N-(3-(benzylthio)propylene)(S)leucine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (R)leucine methyl ester (Procedure 5).

Yield 60% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-65.98, H-8.79, N-4.53; found:C-67.09, H-9.20, N-4.54. (a)_(D23)=−17.4° (C 1.44, DCM).

COMPOUND 48

N-(2-(benzylthio)ethylene)(S)phenylalanine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (R)phenyl lactic acid methyl ester (Procedure 5).

Yield 82% of white crystals. m.p.=48-49° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-69.27, H-7.04,N-4.25; found: C-69.55, H-7.21, N-4.08. (a)_(D14)=−23.3° (C=1.01, DCM).

COMPOUND 49

N-(3-(benzylthio)propylene)(S)phenylalanine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (R)phenyl lactic acid methyl ester (Procedure 5).

Yield 71% of white crystals. m.p.=38-39° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-69.94, H-7.34,N-4.08; found: C-69.66, H-7.39, N-4.37. (a)_(D26)=+2.0° (C 1.00, DCM).

COMPOUND 50

N-(4-(benzylthio)butylene)(S)phenylalanine methyl ester

The title compound was prepared according to procedure 13 from theTriflate of (S)phenyl lactic acid methyl ester (Procedure 5).

Yield 81% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-70.55, H-7.61, N-3.92; found:C-70.51, H-7.69, N-4.22. (a)_(D26)=+4.9° (C 1.00, DCM).

EXAMPLE 14 COMPOUND 51

Boc-N-(2-(benzylthio)ethylene)glycine

The title compound was prepared from Compound 44 by hydrolysis accordingto Procedure 11.

Yield 88% of white crystals. m.p.=71-72° C. NMR (CDCl₃) in agreementwith the title compound. Elemental analysis-calculated: C-59.05, H-7.12,N-4.30; found: C-59.39, H-7.26, N-4.18.

EXAMPLE 15 COMPOUND 52

Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine

The title compound was prepared from Compound 48 by hydrolysis accordingto Procedure 11.

Yield 78% of white crystals. m.p.=82-83° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25)=−105.9° (C 1.01, DCM).

EXAMPLE 16 COMPOUND 53

Boc-N-(3-(benzylthio)propylene)(S)phenylalanine

The title compound was prepared from Compound 49 by hydrolysis accordingto Procedure 11.

Yield 99% of white crystals. m.p.=63-64° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25)=−87.4° (C 1.01, DCM).

EXAMPLE 17 COMPOUND 54

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester(Compound 44) according to Procedure 12.

Yield 32% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:C-64.39, H-7.02, N-5.53. (a)_(D16)=+4.5° (C 0.88, DCM).

EXAMPLE 18 COMPOUND 55

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methylester

Boc-L-Phe was coupled to N-(2-(benzylthio)ethylene) (S)phenylalaninemethyl ester (Compound 48) according to Procedure 12.

Yield 46% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. (a)_(D26)=−115.9° (C 1.0, CHCl₃).

COMPOUND 56

N-Bzl-β-alanine t-butyl ester

A solution of 6.16 g of β-alanine t-butyl ester acetate in 150 mL waterwas reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5% yield

TLC (Method A) Rf=0.78 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 57

N-Bzl-γ-amino butyric acid t-butyl ester

A solution of 6.58 g of γ-aminobutyric acid t-butyl ester acetate in 150mL water was reacted with benzaldhyde (Procedure 2) to give 4.24 g,57.9% yield

TLC (Method A) Rf=0.74 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 58

N^(α)(Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester

A solution of 3.53 g of N-Bzl-β-alanine t-butyl ester (Compound 56) inDMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17). Yield86.9%

TLC (Method F) Rf=0.95 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 59

N^(α)(Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester

A solution of 3.53 g of N-Bzl-γ-aminobutyric acid t-butyl ester(Compound 57) in DMF was reacted with 2.61 mL benzyl bromoacetate(Procedure 17). Yield 83%

TLC (Method F) Rf=0.92 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 60

N^(α)(2-t-butyl carboxy ethylene)glycine

A solution of N^(α)(Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester(Compound 58) in MeOH was hydrogenated (Procedure 8). Yield 87.8%

TLC (Method A) Rf=0.56 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 61

N^(α)(3-t-butyl carboxy propylene)glycine

A solution of N^(α)(Bzl)(3-t-butyl carboxy propylene)glycine benzylester (Compound 59) in MeOH was hydrogenated (Procedure 8). Yield 94%

TLC (Method A) Rf=0.3 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

EXAMPLE 19 COMPOUND 62

N^(α)(Fmoc)(2-t-butyl carboxy ethylene)glycine

A solution of N^(α)(2-t-butyl carboxy ethylene)glycine (Compound 60) inH₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 90%

TLC (Method D) Rf=0.5 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 67.38 6.34 3.11 Calc: 67.75 6.403.29

EXAMPLE 20 COMPOUND 63

N^(α)(Fmoc)(3-t-butyl carboxy propylene)glycine

A solution of N^(α)(3-t-butyl carboxy propylene)glycine (Compound 61) inH₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 82%

TLC (Method D) Rf=0.58 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 68.29 6.83 3.88 Calc: 68.32 6.653.19

COMPOUND 64

(R)-O-Trf-3-Phenyllactic acid benzyl ester

To a cooled solution of Trf₂O and pyridine in dry DCM (Procedure 5), asolution of 5.3 g of (R)-3-Phenyllactic acid benzyl ester was added.After the workup (Procedure 5), the yield was 91.43%. The product wasused immediately or kept in a cold desiccator under Ar.

COMPOUND 65

N^(α)(Bzl)(2-t-butyl carboxy ethylene)(S)Phenylalanine benzyl ester

A solution of 5.48 g of N-Bzl-β-alanine t-butyl ester (Compound 56) inDCM was reacted with 7.35 g of (R)-O-Trf-3-Phenyllactic acid benzylester (COMPOUND 64) in dry DCM (Procedure 6). After workup the crudeproduct was purified by flash chromatography. PE:EtOAc (4:1) 1.5 L.After solvent evaporation under vacuum, the product was dried undervacuum.

Yield 71.5%; TLC (Method C) Rf=0.77 (one spot); (α)_(D)=−62.7 (C=1,MeOH); NMR (CDCl₃) in agreement with the title compound.

COMPOUND 66

N^(α)(2-t-butyl carboxy ethylene)(S)Phenylalanine

A solution of 6.3 g of N^(α)(Bzl)(2-t-butyl carboxyethylene)(S)Phenylalanine benzyl ester (Compound 65) in MeOH washydrogenated (Procedure 8). Yield 48.6%

TLC (Method A) Rf=0.52-0.54 (one spot); NMR (CDCl₃) in agreement withthe title compound.

EXAMPLE 21 COMPOUND 67

N^(α)(Fmoc)(2-t-butyl carboxy ethylene)(S)Phenylalanine

A solution of 2.13 g of N^(α)(2-t-butyl carboxyethylene)(S)Phenylalanine (Compound 66) in H₂O:Et₃N was reacted withFmocOSu (Procedure 9). Yield 38%

TLC (Method D) Rf=0.77 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 71.92 639 2.87 Calc: 72.21 6.452.72

HPLC (Method G) 93%

COMPOUND 68

N^(α)(Bzl)(2-Boc amino ethylene)glycine benzyl ester

Elemental analysis-calculated: C-59.05, H-7.12, N-4.30; found: C-59.39,H-7.26, N-4.18.

EXAMPLE 15 COMPOUND 52

Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine

The title compound was prepared from Compound 48 by hydrolysis accordingto Procedure 11.

Yield 78% of white crystals. m.p.=82-83° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25)=−105.9° (C 1.01, DCM).

EXAMPLE 16 COMPOUND 53

Boc-N-(3-(benzylthio)propylene)(S)phenylalanine

The title compound was prepared from Compound 49 by hydrolysis accordingto Procedure 11.

Yield 99% of white crystals. m.p.=63-64° C. NMR (CDCl₃) in agreementwith the title compound. (a)_(D25)=−87.4° (C 1.01, DCM).

EXAMPLE 17 COMPOUND 54

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester

Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester(Compound 44) according to Procedure 12.

Yield 32% of colorless oil. NMR (CDCl₃) in agreement with the titlecompound. Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:C-64.39, H-7.02, N-5.53. (a)_(D16)=+4.5° (C 0.88, DCM).

EXAMPLE 18 COMPOUND 55

Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methylester NMR (CDCl₃) in agreement with the title compound.

COMPOUND 73

N^(α)(3-Boc amino propylene)glycine

A solution of 0.025 mole of N^(α)(Bzl)(3-Boc amino propylene)glycinebenzyl ester (Compound 69) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 74% of white solid. mp 214-6° C.

TLC (Method A) Rf=0.27 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 74

N^(α)(4-Boc amino butylene)glycine

A solution of 0.025 mole of N^(α)(Bzl)(4-Boc amino butylene)glycinebenzyl ester (Compound 70) in 60 mL MeOH was hydrogenated(Procedure 8).Yield 89.5% of white solid. mp 176-8° C.

TLC (Method A) Rf=0.23 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

COMPOUND 75

N^(α)(6-Boc amino hexylene)glycine

A solution of 0.025 mole of N^(α)(Bzl)(6-Boc amino hexylene)glycinebenzyl ester (Compound 71) in 60 mL MeOH was hydrogenated (Procedure 8).Yield 80% of white solid. mp 172-4° C.

TLC (Method A) Rf=0.26 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

EXAMPLE 22 COMPOUND 76

N^(α)(Fmoc)(2-Boc amino ethylene)glycine

A solution of 0.02 mole of N^(α)(2-Boc amino ethylene)glycine (Compound72) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield 80% ofwhite solid. mp 130-132° C. TLC (Method D) Rf=0.5 (one spot)

NMR (CDCl₃) in agreement with the title compound.

Elemental Analysis: % C % H % N Found: 65.18 6.11 5.91 Calc: 65.43 6.406.63

EXAMPLE 23 COMPOUND 77

N^(α)(Fmoc)(3-Boc amino propylene)glycine

A solution of 0.02 mole of N^(α)(Fmoc)(3-Boc amino propylene)glycine(Compound 73) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield85% of white solid. mp 125° C.

TLC (Method D) Rf=0.5-0.6 (one spot); NMR (CDCl₃) in agreement with thetitle compound.

Elemental Analysis: % C % H % N Found: 66.05 6.65 6.00 Calc: 66.06 6.656.16

EXAMPLE 24 COMPOUND 78

N^(α)(Fmoc)(4-Boc amino butylene)glycine

A solution of 0.02 mole of N^(α)(Fmoc)(4-Boc amino butylene)glycine(Compound 74) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield79.4% of white solid. mp 150-152° C.

TLC (Method D) Rf=0.42-0.47 (one spot); NMR (CDCl₃) in agreement withthe title compound.

Elemental Analysis: % C % H % N Found: 66.35 6.84 5.77 Calc: 66.06 6.885.98

EXAMPLE 25 COMPOUND 79

N^(α)(Fmoc)(6-Boc amino hexylene)glycine

A solution of 0.02 mole of N^(α)(Fmoc)(6-Boc amino hexylene)glycine(Compound 75) in H₂O:Et₃N was reacted with FmocOSu (Procedure 9). Yield81.5% of white solid. mp 78-80° C. TLC (Method D) Rf=0.7 (one spot)

NMR (CDCl₃) in agreement with the title compound.

Elemental Analysis: % C % H % N Found: 68.02 7.08 5.37 Calc: 67.72 7.315.64

SYNTHETIC EXAMPLES

Two series of octapeptide somatostatin analogs of the present inventionwere synthesized, characterized, and tested for biological activity.

1) The first series of compounds corresponds to the general Formula(XIVb); this series comprises compounds of the specific formula

H-(D)Phe-R⁶-Phe-(D)Trp-Lys-Thr-R¹¹-Thr-NH₂

wherein R⁶ and R¹¹ are N^(α) ω-functionalized alkylene amino acidbuilding units.

2) The second series of compounds corresponds to the general Formula(XVIc); this series comprises compounds of the specific formula

H-(D) Phe-R⁶-Phe-(D)Trp-Lys-R¹⁰-Thr-NH₂

wherein R⁶ and R¹⁰ are N^(α) ω-functionalized alkylene amino acidbuilding units.

The structures of these novel synthetic peptide analogs into which N^(α)ω-functionalized amino acid building units were incorporated, aresummarized in Tables 7 and 8. In both series, the building units usedwere glycine building units in which the bridging groups, attached viathe alpha nitrogens to the peptide backbone, were varied.

For the sake of simplicity, these two series are referred to herein asthe SST Gly⁶,Gly¹¹ and SST Gly⁶,Gly¹⁰ series, respectively.

In each series, the position of the cyclization points was constant,while the length and direction of the bridge was varied. Thus, C2,N2refers to a bridge consisting of an amide bond in which the carbonylgroup is closer to the amino end of the peptide and which contains twomethylene groups between the bridge amide and each of the backbonenitrogens involved in the bridge.

Peptide assembly was carried out either manually or with an automaticpeptide synthesizer (Applied Biosystems Model 433A). Following peptideassembly, de-protection of bridging groups that form the cyclizationarms was carried out with Pd(PPh₃)₄ (palladium tetrakis triphenylphosphine) in the case of Allyl/Alloc protecting groups or with TFA inthe case of tBu/Boc protecting groups. For obtaining the linear(non-cyclized) analog, the peptides were cleaved from the resin at thisstage. Cyclization of the peptides was carried out with PyBOP. Cleavageof the peptides from the polymeric support was carried out with suitablereagents depending on the type of resin used, e.g., with TFA for Rinkamide type resins and with HF for mBHA (para-methyl benzhydryl amine)type resins. The crude products were characterized by analytical HPLC.The peptides were purified by preparative reversed phase HPLC. Thepurified products where characterized by analytical HPLC, massspectroscopy, and amino acid analysis.

TABLE 7 SST Gly⁶, Gly¹¹ Example Bridging Compound Crude No. GroupsNumber Method Yield 26 C1,N2 Cyclic DE-3-32-4 1 NA** 27 C1,N2 LinearDE-3-32-2 1 NA 28 C1,N3 Cyclic PTR 3004 2 79 mg 29 C1,N3 Linear PTR 30052 34 mg 30 C2,N2 Cyclic PTR 3002 1 NA 31 C2,N2 Linear PTR 3001 1 NA 32C2,N3 Cyclic PTR 3007 2 40 mg 33 C2,N3 Linear PTR 3008 2 40 mg 34 N2,C2Cyclic YD-9-1661-1 2 NA 35 N2,C2 Linear YD-9-168-1 2 NA 36 N3,C2 CyclicPTR 3010 2 100 mg  37 N3,C2 Linear PTR 3011 2 NA 38 Linear* PTR 3003 396 mg *Linear refers to the identical sequence with Gly residues inplace of R⁶ and R¹¹. **NA denotes not available.

Table 7 methods:

1) Manual synthesis on mBHA resin. HF cleavage.

2) Manual synthesis on Rapp tentagel resin. TFA cleavage.

3) Rink amide resin; assembly in automated peptide synthesizer, 0.1 mmolscale.

TABLE 8 SST Gly⁶, Gly¹⁰ Example Bridging Compound Crude No. GroupsNumber Method Yield 39 C1,N2 Cyclic YD-9-171-3 1 20 mg 40 C1,N2 LinearYD-9-171-2 1 10 mg 41 C1,N3 Cyclic YD-9-175-3 1 44.9 mg 42 C1,N3 LinearYD-9-175-2 1 25.4 mg 43 C2,N2 Cyclic PTR 3019 1 40 mg 44 C2,N2 LinearPTR 3020 1 26 mg 45 C2,N3 Cyclic YD-5-28-3 3 101.5 mg 46 C2,N3 LinearYD-5-28-2 3 48.3 mg 47 N2,C2 Cyclic PTR 3016 2 60 mg 48 N2,C2 Linear PTR3017 2 40 mg 49 N3,C2 Cyclic YS-8-153-1 2 93 mg 50 N3,C2 LinearYS-8-152-1 2 54 mg 51 *Linear PTR 3021 1 100 mg **Acetylated Des-D-Phe⁵52 N3,C2 Cyclic PTR 3013 67 mg 53 N3,C2 Linear PTR 3014 48 mg *Linearrefers to the identical sequence with Gly residues in place of R⁶ andR¹⁰. **Acetylated Des-D-Phe⁵ refers to the same sequence in which the Nterminal D-Phe⁵ is absent and the N-terminus is acetylated.

Table 8 methods:

1) Assembly in automated peptide synthesizer; 0.1 mmol scale. (HBTU).

2) Manual synthesis; PyBrop.

3) Assembly in automated peptide synthesizer, 0.25 mmol scale. (HBTU).

Synthesis of SST Gly⁶,Gly¹⁰ N3,C2:

Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelled inN-methylpyrrolidone (NMP) in a reaction vessel equipped with a sinteredglass bottom and placed on a shaker. The Fmoc protecting group wasremoved from the resin by reaction with 20% piperidine in NMP (2 times10 minutes, 25 ml each). Fmoc removal was monitored by ultravioletabsorption measurement at 290 nm. A coupling cycle was carried out withFmoc-Thr(OtBu)-OH (3 equivalents) PyBrop (3 equivalents) DIEA (6equivalents) in NMP (20 ml) for 2 hours at room temperature. Reactioncompletion was monitored by the qualitative ninhydrin test (Kaisertest). Following coupling, the peptide-resin was washed with NMP (7times with 25 ml NMP, 2 minutes each). Capping was carried out byreaction of the peptide-resin with acetic anhydride (capping mixture:HOBt 400 mg, NMP 20 ml, acetic anhydride 10 ml, DIEA 4.4 ml) for 0.5hours at room temperature. After capping, NMP washes were carried out asabove (7 times, 2 minutes each). Fmoc removal was carried out as above.Fmoc-Phe-OH was coupled in the same manner, and the Fmoc group removed,as above. The peptide resin was reacted with Fmoc-Gly-C2 (Allyl)building unit: coupling conditions were as above. Fmoc removal wascarried out as above. Fmoc-Lys(Boc)-OH was coupled to the peptide resinby reaction with HATU (3 equivalents) and DIEA (6 equivalents) at roomtemperature overnight and then at 50° C. for one hour. Additional DIEAwas added during reaction to maintain a basic medium (as determined bypH paper to be about 9). This coupling was repeated. Coupling completionwas monitored by the Fmoc test (a sample of the peptide resin was takenand weighed, the Fmoc was removed as above, and the ultravioletabsorption was measured). Fmoc-D-Trp-OH was coupled to the peptide resinwith PyBrop, as described above. Following Fmoc removal, Fmoc-Phe-OH wascoupled in the same way. Synthesis was continued with one-fifth of thepeptide resin.

Following Fmoc removal, the second building unit was introduced:Fmoc-Gly-N3(Alloc)-OH by reaction with PYBrop, as described above.Capping was carried out as described above. Following Fmoc removal, thepeptide-resin was divided into two equal portions. Synthesis wascontinued with one of these portions. Boc-D-Phe-OH was coupled byreaction with HATU, as described above for Fmoc-Lys(Boc)-OH. Capping wascarried out as above.

The Allyl and Alloc protecting groups were removed by reaction withPd(PPh₃)₄ and acetic acid 5%, morpholine 2.5% in chloroform, underargon, for 2 hours at room temperature. The peptide resin was washedwith NMP as above. Two-thirds of the resin were taken for cyclization.Cyclization was carried out with PyBOP 3 equivalents, DIEA 6equivalents, in NMP, at room temperature overnight. The peptide resinwas washed and dried. The peptide was cleaved from the resin by reactionwith TFA 81.5%, phenol 5%, water 5%, EDT 2.5%, TIS(tri-isopropyl-silane) 1%, and 5% methylene chloride, at 0° C. for 15minutes and 2 hours at room temperature under argon. The mixture wasfiltered into cold ether (30 ml, 0° C.) and the resin was washed with asmall volume of TFA. The filtrate was placed in a rotary evaporator andall the volatile components were removed. An oily product was obtained.It was triturated with ether and the ether decanted, three times. Awhite powder was obtained. This crude product was dried. The weight ofthe crude product was 93 mg.

PHYSIOLOGICAL EXAMPLES EXAMPLE 54 BRADYKININ ANTAGONIST ASSAY(Displacement of (³H)dopamine release from PC 12 cells)

Novel backbone cyclized peptide analogs of the present invention wereassayed in vitro for bradykinin antagonist activity by protection of(³H)dopamine release from PC 12 cells that express bradykinin receptors.PC12 cells were grown in Dulbecco Modified Eagle's medium with highglucose, supplemented with 10% horse serum, 5% fetal calf serum, 130units/ml penicillin and 0.1 mg/ml streptomycin. For experiments, cellswere removed from the medium using 1 mmole EDTA and replated on collagencoated-12-well plates and assayed 24 hr later. Release of (³H)dopaminewas determined as follows: cells were incubated for 1.5 hr at 37° C.with 0.5 ml of growth medium and 0.85 ml (³H)DA (41 Ci/mmole) and 10mg/ml pargyline followed by extensive washing with medium (3×1 ml) andrelease buffer consisting of (mM): 130 NaCl; 5 KCl; 25 NaHCO₃; 1NaH₂PO₄; 10 glucose and 1.8 CaCl₂. In a typical experiment, cells wereincubated with 0.5 ml buffer for 5 consecutive incubation periods of 3min each at 37° C. Spontaneous (³H)DA release was measured by collectingthe medium released by the cells successively for the first 3 minperiod. Antagonists were added to the cells 3 min prior to stimulation(at the second period), and stimulation of (³H)DA release by 100 nmoleof bradykinin are monitored during the 3 period by 60 mmole KCl. Theremaining of the (³H)DA was extracted from the cells by over nightincubation with 0.5 ml 0.1 N HCl. (³H)DA release during each 3 minperiod was expressed as a % of the total (³H)DA content of the cells.Net evoked release was calculated from (³H)DA release during stimulationperiod after subtracting basal (³H)DA release in the preceding baselineperiod if not indicated otherwise.

At 10⁻⁶ M, Example 1 showed 30% inhibition of BK activity, Example 4showed 17% inhibition of BK activity. Note, the noncyclized (control)peptide of example 2 showed 0% inhibition of BK activity.

EXAMPLE 55 BRADYKININ ANTAGONIST ASSAY (Guinea-pig assay)

The ileum of the guinea-pig was selected as the preparation for thebioassay. This tissue contains predominantly BK₂ receptors. Thepreparation consists of the longitudinal muscle layer with the adheringmesenteric plexus. The isolated preparation was kept in Krebs solutionand contractions were measured with an isometric force transducer. Theguinea-pig ileum is highly sensitive to BK, with EC₅₀ at 2×10⁻⁸ M. Atleast two control responses to BK (2×10⁻⁸ M) were measured previous tomeasuring the responses of backbone cyclized peptides of the presentinvention. Atropine (1 μM) was always present.

At 10⁻⁶ M, Example 1 showed 24% inhibition of BK activity, Example 3showed 10% and Example 4 showed 17% inhibition of BK activity. Note, thenoncyclized (control) peptide of example 2 showed 0% inhibition of BKactivity.

EXAMPLE 56 SOMATOSTATIN ASSAY (Receptor based screening)

Initial screening is conducted using ¹²⁵I-labeled SST analogs andpituitary membrane preparations or cell lines. The binding assay isdescribed in Tran, V. T., Beal, M. F. and Martin, J. B. Science,228:294-495, 1985, which is incorporated herein by reference in itsentirety and is optimized with regards to membrane concentration,temperature and time. The assay is sensitive (nM range) and robust.Selectivity will be based on the recent cloning of the five human SSTreceptors. The ability to screen the compounds with regard to bindingand biological activity in mammalian cells should facilitate thedevelopment of subtype-selective analogs. These compounds are useful inthe treatment of specific endocrine disorders and therefore should bedevoid of unwanted side effects.

EXAMPLE 57 SOMATOSTATIN (SST) ASSAY (In vivo assays)

The biological effects of SST on growth hormone, insulin and glucagonrelease is conducted by measuring the levels of these hormones usingcommercially available RIA test kits. Pharmacological effects of SST inpatients with neuroendocrine tumors of the gut will requiredetermination of 5-hydroxyindole acetic acid (for carcinoid) and VIP(for VIPoma). In vivo visualization of SST receptor-positive tumors isperformed as described by Lambert et al., New England J. Med.,323:1246-1249 1990, following i.v. administration of radio-iodinated SSTanalogs.

EXAMPLE 58 Receptor Binding Specificity of Cyclic Peptide Analogs

Binding of representative peptides of Examples 39-54 to differentsomatostatin receptors was measured in vitro, in Chinese Hamster Ovary(CHO) cells expressing the various receptors. An example of theselectivity obtained with the cyclic peptides is presented in Table 9.The values presented are percent inhibition of radioactive iodinatedsomatostatin (SRIF-14) binding.

TABLE 9 Binding of peptide analogs to somatostatin receptor subtypesSomatostatin Receptor (SSTR) Subtype SSTR 2B SSTR 5 Compound Conc. (M)Description 10⁻⁶ 10⁻⁷ 10⁻⁸ 10⁻⁶ 10⁻⁷ 10⁻⁸ Compound Number PTR 3003Linear 16  3 0 55 20 0 PTR 3004 Cyclic C1,N3 0 0 0 14  0 0 PTR 3005Linear C1,N3 0 0 0  9  0 0 PTR 3007 Cyclic C2,N3 0 0 0 19  9 0 PTR 3008Linear C2,N3 0 0 0 15  6 0 PTR 3010 Cyclic N3,C2 0 0 0 63 26 9 PTR 3011Cyclic N3,C2 0 0 0 27 66 27  Control Peptides BIM 3503 Pos. Control 81 33  16  92 66 27  PTR 4003 Neg. Control 0 0 0  0  0 0

EXAMPLE 59 Resistance to Biodegradation of SST Analogs

The in vitro biostability of a SST cyclic peptide analog, PTR 3002, wasmeasured in human serum, and was compared to the same sequence in anon-cyclic peptide analog (PTR 3001), to octreotide (Sandostatin), andto native somatostatin (SRIF). The results are shown in FIG. 1. In thisassay, the cyclic peptide in accordance with the present invention is asstable as octreotide, is more stable than the corresponding non-cyclicstructure, and is much more stable than SRIF. The assay was based onHPLC determination of peptide degradation as a function of time at 37°C.

EXAMPLE 60 Inhibition of Growth Hormone Release by SST Analogs

In vivo determination of the pharmacodynamic properties of cyclicpeptide analogs was carried out. Inhibition of Growth Hormone (GH)release as a result of peptide administration was measured. Measurementswere carried out in Sprague-Dawley male rats: peptide analog activitywas compared in this study to SRIF or to octreotide (Sandostatin). Eachgroup consisted of 4 rats. Time course profiles for GH release underconstant experimental conditions were measured.

Methods

Adult male Sprague-Dawley rats, specific pathogen free (SPF), weighing200-350 g, were maintained on a constant light-dark cycle (light from8:00 to 20:00 h), temperature (21±3° C.), and relative humidity(55±10%). Laboratory chow and tap water were available ad libitum. Onthe day of the experiment, rats were anesthetized with pentobarbitone(50 mg/kg). Rats anesthetized with pentobarbitone exhibit lowsomatostatin levels in portal blood vessels. (Plotsky, P. M., Science,230, 461-463, 1985). A single blood sample (0.6 ml) was taken from theexposed cannulated jugular vein for the determination of the basal GHlevels (−15 min). Immediately thereafter the appropriate peptidepretreatment was administered. The animals received 10 μg/kg of eithernative somatostatin (SRIF) or the synthetic analog octreotide(Sandostatin), or the cyclic peptide analog. A saline solution (0.9%NaCl) was administered as a control. All peptides were administeredsubcutaneously in a final volume of 0.2 ml. Further sampling was carriedout at 15, 30, 60, and 90 minutes after peptide administration.Immediately after the collection of each blood sample, an appropriatevolume (0.6 ml) of saline was administered intravenously. Blood sampleswere collected into tubes containing heparin (15 unites per ml of blood)and centrifuged immediately. Plasma was separated and kept frozen at−20° C. until assayed.

Rat growth hormone (rGH) [¹²⁵I] levels were determined by appropriateradioimmunoassay kit (Amersham). The standard in this kit has beencalibrated against a reference standard preparation (NIH-RP2) obtainedfrom the National Institute of Diabetes and Digestive and KidneyDiseases. All samples were measured in duplicate.

EXAMPLE 61 Lack of Toxicity of Cyclized Peptide Analogs

PTR 3007 at a dose of 1.5 mg/kg was well tolerated after singleintraperitoneal application. PTR 3013 was not toxic to the rats evenwith doses of 4 mg/kg. These two doses are several orders of magnitudehigher than those needed to elicit the desired endocrine effect. Thepeptides dissolved in saline produced no untoward side effects on thecentral nervous system, cardiovascular system, body temperature, nor onthe periphery of the animals. Rats were observed for 4 hours postadministration of the peptides. PTR 3007 and 3013 produced norespiratory disturbances, did not result in the appearance ofstereotyped behavior, or produce any changes in muscle tone. After 3hours, postmortem examination did not detect any abnormality in theliver, kidneys, arteries and veins, gastrointestinal tract, lungs,genital system, nor the spleen.

2 14 amino acids amino acid single linear peptide not provided 1 Ala GlyCys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 1 5 10 9 amino acidsamino acid single linear peptide not provided 2 Arg Pro Pro Gly Phe SerPro Phe Arg 1 5

What is claimed is:
 1. A backbone cyclized peptide analog having thegeneral Formula (I):

wherein: a and b each independently designates an integer from 1 to 8 orzero; d, e, and f each independently designates an integer from 1 to 10;(AA) designates an amino acid residue wherein the amino acid residues ineach chain may be the same or different; E represents a hydroxyl group,a carboxyl protecting group or an amino group, or CO—E can be reduced toCH₂—OH; each of R, R′, R″, and R′″ is independently hydrogen or an aminoacid side-chain optionally bound with a specific protecting group; andthe lines designate a bridging group of the Formula: (i) —X—M—Y—W—Z—; or(ii) —X—M—Z— wherein: one line may be absent; M and W are independentlyselected from the group consisting of disulfide, amide, thioether,thioester, imine, ether, and alkene; and X, Y and Z are eachindependently selected from the group consisting of alkylene,substituted alkylene, arylene, homo- or hetero-cycloalkylene andsubstituted cycloalkylene.
 2. The backbone cyclized peptide analog ofclaim 1 wherein —X—M—Y—W—Z— is: —(CH₂)_(x)—M—(CH₂)_(y)—W—(CH₂)_(z)—wherein M and W are independently selected from the group consisting ofdisulfide, amide, thioether, thioester, imine, ether, and alkene; x andz each independently designates an integer of from 1 to 10, and y iszero or an integer of from 1 to 8, with the proviso that if y is zero, Wis absent.
 3. The backbone cyclized peptide analog of claim 1 whereinthe group CO—E is CH₂OH.
 4. The backbone cyclized peptide analog ofclaim 1 wherein R is CH₃—, (CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—,CH₃S(CH₂)₂—, HOCH₂—, CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—,NH₂(CH₂)₃—, HOC(═O)CH₂—, HOC(═O)(CH₂)₂—, NH₂(CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—,HO-phenyl-CH₂—, benzyl, methylindole, or methylimidazole.
 5. Thebackbone cyclized peptide analog of claim 1 wherein R′ is CH₃,(CH₃)₂CH—, (CH₃)₂CHCH₂—, CH₃CH₂CH(CH₃)—, CH₃S(CH₂)₂—, HOCH₂—,CH₃CH(OH)—, HSCH₂—, NH₂C(═O)CH₂—, NH₂C(═O)(CH₂)₂—, C(NH₂)₂ NH(CH₂)₃—,HOC(═O)CH₂—, HOC(═)(CH₂—, NH₂(CH₂)₄—, C(NH₂)₂ NH(CH₂)₃—, HO-phenyl-CH₂—,benzyl, methylindole, or methylimidazole.
 6. A backbone cyclized peptideanalog having the general Formula (XIVa):

wherein m and n are 1, 2 or 3; X is CH₂OH or NH₂; R⁵ is absent or isGly, (D)- or (L)-Ala, Phe, Nal, and β-Asp(Ind); R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R⁷ is Phe or Tyr; R¹⁰ to is absentor is Gly, Abu, Thr or Val; R¹² is absent or is Thr or Nal; and Y² isselected from the group consisting of amide, disulfide, thioether,imine, ether, and alkene.
 7. A backbone cyclized peptide analog havingthe general Formula (XIVb):

wherein m and n are 1, 2 or 3; X is CH₂OH or NH₂; R⁶ and R¹¹ areindependently Gly or (D)- or (L)-Phe; R⁷ is Phe or Tyr; R¹⁰ is absent oris Gly, Abu, Thr or Val; and Y² is selected from the group consisting ofamide, disulfide, thioether, imine, ether, and alkene.
 8. A backbonecyclized peptide analog having the general Formula (XVa):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or (L)-Phe;R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or (L)-Phe; R¹² isabsent or is Thr or Nal, and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imine, ether, and alkene.
 9. A backbonecyclized peptide analog having the general Formula (XVb):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁶ is(D) or (L)-Phe; R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or(L)-Phe; R¹² is absent or is Thr or Nal, and Y¹ is selected from thegroup consisting of amide, disulfide, thioether, imine, ether, andalkene.
 10. A backbone cyclized peptide analog having the generalFormula (XVIa):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or (L)-Phe;R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or (L)-Phe; R¹² isabsent or is Thr or Nal, and Y¹ is selected from the group consisting ofamide, disulfide, thioether, imine, ether, and alkene.
 11. A backbonecyclized peptide analog having the general Formula (XVIb):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁶ is(D) or (L)-Phe; R¹⁰ is absent or is Gly, Abu or Thr; and R¹¹ is (D)- or(L)-Phe; R¹² is absent or is Thr or Nal, and Y¹ is selected from thegroup consisting of amide, disulfide, thioether, imine, ether, andalkene.
 12. A backbone cyclized peptide analog having the generalFormula (XVIc):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R⁶ is (D) or (L)-Phe;and R¹⁰ is absent or is Gly, Abu or Thr; R¹² is absent or is Thr or Nal,and Y¹ is selected from the group consisting of amide, disulfide,thioether, imine, ether, and alkene.
 13. The backbone cyclized peptideanalog of claim 1 having the general Formula (XIXa):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R¹⁰ is absent or isGly, Abu or Thr; R¹² is absent or is Thr or Nal; and Y¹ is selected fromthe group consisting of amide, disulfide, thioether, imine, ether, andalkene.
 14. The backbone cyclized peptide analog of claim 1 having thegeneral Formula (XIXb):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; and Y¹is selected from the group consisting of amide, disulfide, thioether,imine, ether, and alkene.
 15. The backbone cyclized peptide analog ofclaim 1 having the general Formula (XXa):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R⁵ isabsent or is (D)- or (L)-Phe, Nal, or β-Asp(Ind); R¹⁰ is absent or isGly, Abu or Thr; R¹² is absent or is Thr or Nal; and Y¹ is selected fromthe group consisting of amide, disulfide, thioether, imine, ether, andalkene.
 16. The backbone cyclized peptide analog of claim 1 having thegeneral Formula (XXb):

wherein i and j are independently 1, 2 or 3; X is CH₂OH or NH₂; R¹⁰ isabsent or is Gly, Abu or Thr; R¹² is absent or is Thr or Nal; and Y¹ isselected from the group consisting of amide, disulfide, thioether,imine, ether, and alkene.
 17. A pharmaceutical composition comprisingthe backbone cyclized peptide analog of claim 1 and a pharmaceuticallyacceptable carrier or diluent.